1

Inhibition of Gut-derived Lipopolysaccharide-induced Inflammatory

Pathways as a Treatment Strategy for Nonalcoholic steatohepatitis

1 Zhijia Zhou1†, Huiqing Liang 2†, Jing Xu 3†, Lingxia Xu1,4†, Shaoliang Zhang1, Shilin Xu1, Ning
2 Qu1, Yaoyao Wang1, Limin Zhang1, Yanmiao Yang1, Xiaoying Li1, Penghua Lai1* and
3 Shaodong Chen1*

4 1
Department of Traditional Chinese Medicine, School of Medicine, Xiamen University, Fujian
5 361102, Fujian Province, China;

6 2
Center for Liver Disease, Xiamen Hospital of Traditional Chinese Medicine, Fujian 361009, Fujian
7 Province, China;

8 3
College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian Province,361102, China

9 4
Department of Rheumatology, Shanghai Guanghua Hospital, Shanghai University of Traditional
10 Chinese Medicine, Shanghai 201203, China;

11 †These authors have contributed equally to this work and share first authorship

12 * Correspondence:
13 Penghua Lai and Shaodong Chen
14 email: adong@xmu.edu.cn

15 Keywords: Treatment strategy; Pioglitazone; Probiotic; Chlorogenic Acid and Geniposide;

16 Nonalcoholic steatohepatitis; Inflammation; Lipopolysaccharide

17 Abstract

18 Nonalcoholic steatohepatitis (NASH) is associated with high mortality and markedly poor prognosis.
19 Due to the lack of effective target or strategy for drug design, the mainstay of NASH treatment
20 remains weight reduction. However, pioglitazone (PH) has been confirmed potently ameliorating
21 NASH while also increasing body weight. Accordingly, we hypothesized that for nonalcoholic fatty
22 liver disease to NASH transition, gut-derived lipopolysaccharide (LPS) is the dominant trigger rather
23 than lipid accumulation. In this study, we used three drugs including Chlorogenic Acid and
24 Geniposide (CG), Pioglitazone and Bifico (a type of probiotic mixture containing Bifidobacterium,
2 Running Title

25 Lactobacillus acidophilus, and Enterococcus faecalis) in the NASH rat model. We comprehensively
26 studied the LPS-induced inflammatory pathway activation in NASH and validate the assumption.
27 Our results showed all drugs help improve NASH with modulation of intestinal microflora, repairing
28 the intestinal barrier and weight gain. In contrast to earlier studies, probiotic administration resulted
29 in a considerable gain in body weight. In addition, we found that CG reduces amounts of LPS and
30 lipid deposition the most. Therefore, reduction of LPS may be an effective treatment strategy for
31 NASH.

32 1 Introduction

33 Nonalcoholic steatohepatitis (NASH), an extreme form of nonalcoholic fatty liver disease (NAFLD),
34 is defined by a conjunction of steatosis and inflammation, which presents with or without fibrosis[1].
35 NASH is a serious risk factor for cardiovascular disease, portal hypertension, type 2 diabetes mellitus
36 (T2DM) and severe kidney diseases[2] ， and will eventually lead to end-stage liver disease like
37 cirrhosis and hepatocellular carcinoma[3].Thus, it is associated with high mortality and markedly
38 poor prognosis. Currently, the therapeutic options for NASH are limited with no US Food and Drug
39 Administration (FDA) -approved pharmacotherapy. However, the changes in life style (including diet
40 and exercise) aiming to reduce fatty liver by body weight (BW) loss is recommended[4], and several
41 drugs have been shown to be effective in an animal model of NASH.
42 Studies have shown that innate immunity plays an essential role in the pathogenesis of NASH[5].
43 Lipopolysaccharide (LPS) induces innate immune responses by activating Toll-like receptor 4
44 (TLR4) in hepatocytes, producing inflammatory cytokines (including tumor necrosis factor-α, IL-1,
45 IL-6, and chemokines), which leads to inflammatory response and liver injury[6-10]. It is now
46 commonly established that several nutrients known to induce NAFLD alter the gut microbiota and
47 cause dysbiosis (a change in gut microbiota composition), which increasing the permeability of the
48 gut barrier and facilitating translocation of gut microbe–derived products, such as lipopolysaccharide
49 (LPS), to the liver through the portal circulation.[7, 11]A meta-analysis indicated that, compared with
50 healthy volunteers, patients with NASH were more likely to have enhanced intestinal
51 permeability[12] LPS-binding protein (LBP), which is produced mostly by the liver and helps
52 mediate the LPS-induced inflammatory response[13]. Exposure of the liver to LPS caused by
53 increased intestinal permeability therefore predisposes the liver to inflammatory damage. Prior
54 studies have found that higher levels of intestinal LPS is noted in NASH patients than in healthy
55 subjects.[14] Mice lacking MyD88 have been shown to be unable to produce TNF, IL-1β or IL-6 in
56 response to LPS [15] LPS sensing by TLR4 at the plasma membrane promotes the MyD88-

3 2
4 Running Title

57 dependent rapid activation of two major transcription factors implicated in the regulation of
58 inflammation (AP-1 and NF-ĸB) [16] Therefore, regulating gut microbiota to suppress LPS-mediated
59 inflammatory responses have become a new research direction for the treatment of NASH.
60 Based on accumulating evidence, pioglitazone (PH) has been considered the most promising
61 candidates in drug development to protect against NASH. Clinical evidence supported the use of PH
62 as a pharmacological agent to manage NASH[17, 18]. PH significantly improved hepatic steatosis
63 and inflammation but trigger serious side effects such as formation of edema and weight gain[19].
64 The side effects may imply that the reduction of hepatic lipid accumulation is not the first choice of
65 treatment for NASH. BIFICO capsules contain a mixture of Bifidobacterium, Lactobacillus
66 acidophilus, and Enterococcus faecalis and were approved as an over-the-counter (OTC) product by
67 the State Food and Drug Administration (SFDA), in October 2002[20]. Previously studies
68 demonstrated that BIFICO treatment could ameliorate H. pylori-induced gastritis by inhibiting TLR
69 activation[21]. Meanwhile, BIFICO can regulate the status of intestinal flora[22, 23], suggesting that
70 it may play a role in LPS-stimulated flora dysbiosis.
71 A shortfall in the number of effective drugs make both patients and physicians have recently turned
72 their attention to the natural remedies found in Eastern medicine.[24-27]. Geniposide is an iridoid
73 glycoside, abundantly available in the herb Gardenia jasminoides, and is notable for treating various
74 diseases with inflammation or oxidative stress, including brain depression, cancer and diabetes[28-
75 31]. A recent study showed that gardenia glycosides can inhibit the TLR4/ MyD88 pathway to exert
76 anti-tumour effects[29]. Chlorogenic acid (CGA) is a kind of traditional Chinese medicine, abundant
77 in honeysuckle and eucommia, and has a wide range of biological activities, and pharmacological
78 effects[32-34]. Previous studies have shown that co-administration with CGA improved the
79 inflammation-lowering effects of curcumin, Low-dose combination of complimentary bioactives
80 appears to be an attractive strategy for limiting barriers to efficacy of bioactive compounds[35]. In
81 fact, some unexpected results can be achieved with the combination of small molecule drugs[30, 36,
82 37].however, The combination of Geniposide and Chlorogenic acid, PH and BIFICO in a high-fat
83 diet-induced rat model has not been elucidated.
84 In the current study, we hypothesized that inhibition of LPS-induced inflammatory can effectively
85 stop the progression of NAFLD to NASH and evaluate the role of LPS in NAFLD to NASH
86 transition by using PH, BIFICO and CG in the NASH rat model.

87 2 Material and methods

5 3
 6 Running Title

88 2.1 Plant materials and standards

89 Chlorogenic acid (IUPAC name: (1S,3R,4R,5R)3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-

90 1,4,5trihydroxycyclohexane-1-carboxylic acid, purity >98%) and Geniposide (IUPAC name: methyl
91 (1S,4aS,7aS)7-(hydroxymethyl)-1-[(2S,3R,4S,5S,6R)-3,4,5trihydroxy-6-(hydroxymethyl)oxan-2-
92 yl]oxy-1,4a,5,7atetrahydrocyclopenta[c]pyran-4-carboxylate, purity >98%) are commercial products
93 of Nanjing ZeLang Biological Technology Co., Ltd(ZL20170409528). pioglitazone hydrochloride
94 was purchased from Chongqing Kerui Pharmaceutical (Group) Co., Ltd(H20080271) and bifico was
95 purchased from Shanghai shangyaoxinyi Pharmaceutical Co., Ltd(S10950032).

96 2.2 Animals and treatment

97 Sprague–Dawley (SD) rats (body weight 200 ± 20 g) were purchased from the Shanghai Sippe-Bk
98 Lab Animal Co., Ltd. All rats were housed and kept under controlled conditions (23 ± 2°C), humidity
99 (60 ± 10%) and a 12-hour light-dark cycle. The rats adapted to the conditions for a week and were
100 then randomly allocated into a high-fat-diet (HFD) model group and normal diet control group (C
101 group; n=6). Rats were fed according to a high-fat diet (77.5% common feed + 0.5% sodium cholate
102 + 2% cholesterol + 5% soya bean + 5% sucrose + 10% lard from) for 16 weeks in order to produce a
103 NAFLD rat model [26]. Experiments were conducted following established animal protocols and the
104 guidelines approved by the animal experimental ethics committee of Xiamen University (approval
105 No. XMULAC20200055).

106 At the beginning of the 9th week, the rats in the HFD-fed model group were randomly divided into 4
107 groups (n=6 in each group): Model group (M group), Chlorogenic acid and Geniposide group (CG
108 group), Pioglitazone hydrochloride group (PH group) and Bifid triple viable capsule (Bifico) group
109 (B group). The groups mentioned above were fed a HFD for another 8 weeks. The rats in the C group
110 were fed with common feed for 16 weeks. From the 9th week, rats in the M and C groups were fed
111 with double distilled water in the dose of 10 ml/kg-day absorbed intact in the stomach. Rats in the
112 CG group were fed with chlorogenic acid and geniposide aborbed intact in the stomach. Rats in the
113 CG group were also fed a HFD for 16 weeks, followed by a daily gavage of a mixture of a dosage of
114 60 mg/kg geniposide and a dose of 60 mg/kg-day chlorogenic acid (weight ratio=66.7:1) in addition
115 to a HFD for an additional 8 weeks. Rats in the PH group were fed with pioglitazone hydrochloride
116 in the dose of 10 mg/kg-day absorbed intact in the stomach. Rats in the B group were fed with bifico
117 in the dose of 210 mg/kg-day absorbed intact in the stomach.

7 4
 8 Running Title

118 2.3 Sample collection

119 At the end of the 16th week, blood was extracted from the abdominal aorta in all the rats and the
120 body weight was weighed. Euthanasia of the rats was carried out by cervical dislocation according to
121 the AVMA Guidelines on Euthanasia and then liver and epididymal adipose tissue samples were
122 weighed and collected. Specifically, the rats were given an intraperitoneal injection of 1% sodium
123 pentobarbital solution at a dose of 40 mg/kg before being sacrificed for cervical dislocation. the
124 thumb and index finger are placed on either side of the neck at the base of the rats’ skull. With the
125 other hand, the base of the tail or the hind limbs are quickly pulled, causing separation of the cervical
126 vertebrae from the skull. Fecal samples were collected immediately for gut microbiota analysis. The
127 liver and terminal ileum samples were stored in the 10% formaldehyde for histopathological analysis.
128 The rest of the liver samples were stored at -80°C condition for western blotting and biochemical
129 analysis.

130 2.4 Determination of AST, ALT in serum

131 Blood samples were collected from the abdominal aorta and stored at room temperature (RT) for 1 h.
132 After centrifugation at 3500 rpm for 10 min, serum samples were collected for determination of ALT
133 and AST levels using commercial kits (Nanjing Jiancheng Bioengineering Institute).

134 2.5 Histopathological evaluation of liver and the terminal ileum tissue

135 Formalin-fixed and paraffin-embedded the liver and ileum tissue, stained with hematoxylin-eosin
136 (HE), Immunohistochemistry of ileum sections were incubated with antibodies against zonula
137 occludens 1 (ZO-1) (1:100; GB111402, Servicebio, Wuhan, China) and Occludin (1:100; GB111401,
138 Servicebio, Wuhan, China). Livers embedded in an optimum cutting temperature compound were
139 used for oil red O staining for assessment of hepatic steatosis. The procedure was performed as
140 previously detailed and observed under an optical microscope (magnification 400×). All tissues were
141 evaluated by two experienced and ‘blinded’ pathologists and the histological scoring system for
142 Nonalcoholic steatohepatitis (NASH) was conducted according to the NAS score system:0=basically
143 no inflammation, 1≤400-fold field of view, 2=400–200-fold field of view, 3⩾200–100-fold field of
144 view, 4=up to the 40-fold field of view. Color thresholding and measurement of area fraction with
145 ImageJ was also utilized (National Institutes of Health, Bethesda, MD).

146 2.6 Determination of triglyceride (TG) and cholesterol (CHO) in liver

9 5
 10 Running Title

147 100mg of liver samples were weighed and then 0.9mL of absolute ethanol was added in to the
148 centrifuge tube. Afterwards, the liver samples were homogenized with an electric homogenizer upon
149 ice water and centrifuged at 4°C 2500 r/min for 10 min. 2.5 μl of supernatant for TG determination
150 was acquired. The TG content was determined by using a commercial kit (Nanjing Jiancheng
151 Bioengineering Institute) and the cholesterol was measured by a cholesterol assay kit (Nanjing
152 Jiancheng Bioengineering Institute). The final concentrations of triglycerides and cholesterol were
153 corrected for protein content.

154 2.7 DNA Amplification and High-Throughput Sequencing

155 100mg of liver samples were weighed and then 0.9mL of absolute ethanol was added in to the
156 centrifuge tube. Afterwards, the liver samples were homogenized with an electric homogenizer upon
157 ice water and centrifuged at 4°C 2500 r/min for 10 min. 2.5 μl of supernatant for TG determination
158 was acquired. The TG content was determined by using a commercial kit (Nanjing Jiancheng
159 Bioengineering Institute) and the cholesterol was measured by a cholesterol assay kit (Nanjing
160 Jiancheng Bioengineering Institute). The final concentrations of triglycerides and cholesterol were
161 corrected for protein content.

162 2.8 DNA Amplification and High-Throughput Sequencing

163 100mg of liver samples were weighed and then 0.9mL of absolute ethanol was added in to the
164 centrifuge tube. Afterwards, the liver samples were homogenized with an electric homogenizer upon
165 ice water and centrifuged at 4°C 2500 r/min for 10 min. 2.5 μl of supernatant for TG determination
166 was acquired. The TG content was determined by using a commercial kit (Nanjing Jiancheng
167 Bioengineering Institute) and the cholesterol was measured by a cholesterol assay kit (Nanjing
168 Jiancheng Bioengineering Institute). The final concentrations of triglycerides and cholesterol were
169 corrected for protein content.

170 2.9 Western Blotting

171 The liver tissue was lysed in RIPA with 1% PMSF and the protein was then fractionated by
172 electrophoresis on 10% SDS-PAGE. Then, the protein was transferred to the PVDF membrane.
173 Western blotting was used to determine the expression of TLR4 (Proteintech Group, Chicago, IL,
174 USA), MYD88 (Proteintech Group, Chicago, IL, USA) and AP-1(Proteintech Group, Chicago, IL,
175 USA), with GAPDH Antibody (Proteintech Group, Chicago, IL, USA) as the internal reference. The

11 6
 12 Running Title

176 ratio of phosphorylated NF-κB p65 protein (Cell Signaling Technology, Danvers, MA, USA) to total
177 NF-κB p65 protein (Proteintech Group, Chicago, IL, USA) content indicated its phosphorylation
178 level. The antibody-antigen complexes were visualized by employing the ECL
179 (Electrochemiluminescence) kit.

180 2.10 Determination of inflammatory cytokines (IL-1β, IL-6, TNF-α) in liver and plasma

181 100 mg of liver samples were weighed and then 0.9 mL of physiological saline was added into the
182 centrifuge tube. Afterwards, the liver tissue was homogenized with an electric homogenizer upon ice
183 water and centrifuged at 4°C 2500 r/min for 20 min. 10 μl of supernatant was taken for
184 corresponding inflammatory cytokines (IL-1β, IL-6, TNF-α) and determined each time. The
185 inflammatory cytokines (IL-1β, IL-6, TNF-α) content were determined by specific ELISA kits
186 according to the manufacturer's instructions (Beijing Winter Song Boye Biotechnology Co. Ltd.). IL-
187 1 β , IL-6, and TNF-α in plasma were measured by enzyme-linked immunosorbent assay (ELISA),
188 according to the recommended procedures on the kits (IL-1 β, lot No. A301B00631 MULTI
189 Sciences; IL-6, lot No. A30600115 MULTI Sciences; TNF-α, lot No. 222131-001, invitrogen.,
190 USA).

191 2.11 Statistical analysis

192 Data were analyzed by using SPSS 21.0 software and data that conformed to the normal distribution
193 were expressed as the mean ± SD. T-test was used for comparison between two groups, and one-way
194 analysis of variance was utilized for comparison among multiple groups. P-value <0.05 was
195 significantly different.

196 3 Result

197 3.1 Drug treatments attenuated liver injury with gained weight

198 After the 16 week experiment, rats in the M group gained more epididymal fat weight than the
199 control group, while the CG group gained less compared with the M group, but there was no
200 significant difference between the PH and B groups (Fig 1A, B). The liver wet weight and hepatic
201 index were significantly increased in the M group, respectively, compared with the C group. Post
202 intervention at 8-weeks, the liver wet weight and hepatic index in the CG and PH group were
203 significantly decreased compared to those in the M group, however, there was no significant
204 difference between the B and M groups (Fig. 1C, D). The serum levels of ALT and AST were

13 7
 14 Running Title

205 significantly increased in the M group compared with the control group and significantly decreased
206 after the drugs intervention at 8-weeks (Fig. 1E, F).

207

208 Fig. 1 Effect of drug treatments on body weight, obesity and serum transaminases. A. Body weight changes of rats. B. Fat
209 weight of rats in epididymal tissue at the 16th week. C. The liver wet weight of rats at week 16. D. Hepatic index (hepatic
210 index = liver weight/body weight × 100) of rats. The effect of drugs on the liver injury was determined by detecting the
211 serum levels of ALT(E) and AST(F). Statistical analyses were conducted with one-way ANOVA. #P < 0.05, ##P<0.01, ###P
212 < 0.001 versus C; *P < 0.05, **P < 0.01, ***P < 0.001 versus model group.

213 3.2 Drug treatments profoundly changed the composition of the intestinal microbiota in
214 HFD-fed rats

215 To reveal the effects of HFD and the administration of drugs on the microbiota structure, we
216 sequenced the bacterial 16S rRNA at baseline and after 16 weeks, OTU levels by Shannon diversity
217 index were significantly increased in the CG, PH and B groups compared with the M group (Fig.2A).
218 Unweighted PCoA, PCoA and NMDS analyses were conducted to provide an overview of the gut
219 microbiota composition of the five animal groups at baseline and the end of the trial. The plotted
220 scores showed a substantial change in gut microbiota composition in rats fed an HFD. The results
221 indicated that the HFD-induced NASH model had a considerable impact on the gut microbiota
222 composition. CG intervention shifted the overall structure of the HFD-disrupted gut microbiota
223 toward that of the control rats (Fig. 2B–D). Venn diagrams also showed that 377 OTUs were shared
224 between the C and CG groups, 277 OTUs were shared between the C and M groups (Fig. 2E). A

15 8
 16 Running Title

225 hierarchical cluster analysis (HCA) was conducted, generating a heatmap that provided a
226 comprehensive overview of which groups were clustered. The heatmap showed that the distance
227 between the C and CG groups was closer than C and M’s (Fig. 2F). 16 weeks of HFD feeding
228 induced extensive changes in the gut microbial community structure at the phylum level compared
229 with the C group. There was an increase in the abundance of Firmicutes (95.0% vs. 82.4%) and
230 decreases in the abundances of Actinobacteria (0.03% vs. 0.13%), Fusobacteria (0.007% vs. 0.002%)
231 and Proteobacteria (0.1% vs. 0.3%). However, CG intervention mitigated the HFD-induced decrease
232 in Actinobacteria and Proteobacteria, and the HFD-induced increase in Firmicutes. At the genus
233 level, Streptococcus, Staphylococcus, Corynebacterium_1 were decreased in the M group compared
234 to the C group, all of which were reversed by CG intervention. CG intervention decreased the
235 abundances of Lactobacillus and Romboutsia compared with the HFD group (Fig. 2G, H). The plot
236 showed that the abundance of Firmicutes, Actinobacteria and Fusobacteria had a significant
237 difference in five groups (Fig.2I). In our experiment, the abundance of Firmicutes was much higher
238 in the M group compared to the C group, and reversed by CG, PH and B intervention (Fig. 2J).
239 However, the abundance of Bacteroidetes was similar among the five groups (Fig.2K). The ratio of
240 Firmicutes/Bacteroidetes (F/B) was also compared as a featured sign of obesity among the five
241 groups. As indicated in Figure 2L, HFD significantly increased the ratio of F/B, and its effect can be
242 conversed by CG and B.

17 9
 18 Running Title

243

244 Fig. 2 Effects of drugs administration on intestinal microbiota. (A) OTU levels by Shannon diversity index in each group. (B,
245 C) Scatter plots of the unweighted-UniFrac principal coordinates analysis (PCOA) score showing the similarity of the 30
246 bacterial communities based on the UniFrac distance and Scatter plot of the weighted-PCOA score. (D) Nonmetric
247 multidimensional scaling (NMDS) showing the difference in bacterial communities according to the Bray-Curtis distance. (E)
248 Venn diagram representing the number of OTUs among the C, M and CG groups. (F) Heatmap of the hierarchical cluster
249 analysis. (G, H) The bacterial composition of the different communities at the genus level (G) and phylum level (H). (I)
250 Details of the differences in the genus level of the microbiota in each group. The relative abundance of firmicutes (J) and
251 Bacteroidetes(K) in each group. (L) The ratio of Firmicutes/Bacteroidetes. Statistical analyses were conducted with one-way
252 ANOVA. #P < 0.05, ##P<0.01, ###P < 0.001 versus C; *P < 0.05, **P < 0.01, ***P < 0.001 versus model group.

19 10
 20 Running Title

253 3.3 Terminal ileum epithelial barrier damage is diminished by administration of drugs in rats
254 fed an HFD, leading to the inhibition of LPS leakage

255 The HE staining demonstrated that the HFD-induced terminal ileum injury was alleviated by drugs
256 intervention (Fig.3A). The terminal ileum tissue of the rats in the C group was structurally intact, and
257 the intestinal villi were neat and had an orderly arrangement. The terminal ileum tissue of rats in the
258 M group presented damage to the intestinal mucosal barrier, the intestinal epithelial cells were
259 detached, and the intestinal mucosal mechanical barrier was impaired. After treatment with CG, the
260 ileal mucosal structure fully recovered, and the propria intestinal gland was abundant with goblet
261 cells. After treatment with PH, the intestinal villi were orderly arranged, and there was a small
262 amount of intestinal epithelial cells detached. After treatment with B, an intact terminal ileum
263 structure, rich intestinal gland, and abundance of goblet cells can be observed. The expression levels
264 of tight junction proteins were also examined, such as Occludin and zonula occluden-1 (ZO-1). The
265 protein levels of Occludin and ZO-1were reduced in the M group, compared to the C group, and CG
266 and B treatment rescued the protein expression of Occludin (Figure 3B, D) and ZO-1(Figure 3C,E)
267 through Immunohistochemistry. The plasma level of endotoxin was determined, which is an LPS and
268 one of the main components of the cell wall of gram-negative bacteria. With the increase of gram-
269 negative intestinal bacteria in the M group, the LPS content in the plasma of M group was
270 significantly increased compared with that in the C group. After treatment, the LPS content in the
271 plasma was significantly decreased in the CG group, but not in the PH group and B group (Fig. 3F).
272 From these results it can be observed that CG can prevent leaking of endotoxin from the gut into the
273 bloodstream by upregulating the expression of TJ proteins.

21 11
 22 Running Title

274

275 Fig 3. Beneficial effects of drugs on the terminal ileum and the level of serum LPS. (A) Formalin-fixed and paraffin-embedded terminal
276 ileum of the C, M, CG, PH and B groups were stained with hematoxylin & eosin (HE) and observed under an optical microscope (HE,
277 ×400), the yellow arrow marks the shed intestinal epithelial cells. (B) Representative immunostaining of Occludin (B) and zonula
278 occluden-1 (ZO-1) (C), respectively. (D, E) The expression of Occludin (D) and ZO-1(E) of terminal ileum analyzed with ImageJ (F) the
279 LPS content in plasma. Statistical analyses were conducted with one-way ANOVA. #P < 0.05, ##P<0.01, ###P < 0.001 versus C; *P <
280 0.05, **P < 0.01, ***P < 0.001 versus model group.

281 3.4 Hepatic inflammation and hepatic steatosis are attenuated by drug treatments in rats fed
282 an HFD

283 Hepatic steatosis is a predominant feature of NAFLD. For this reason, examined the effect of drugs
284 on NASH induced by the HFD diet was examined. As shown in Fig. 4A, in contrast to the C group,
285 there was extensive steatosis and ballooning of hepatocytes and partial hepatocyte necrosis with a
286 large area of nuclear fragmentation or dissolution with inflammation in the M group by H&E.
287 Notably, HFD-induced steatosis and ballooning in the liver were decreased by Drug treatments (Fig.
288 4A). The oil red O staining also demonstrated that HFD-induced lipid droplets in the liver were

23 12
 24 Running Title

289 reduced by Drug treatments (Fig. 4B, C). The TG content was significantly increased in the liver
290 tissue of the M group compared with that of the C group. After treatment, the TG content in the liver
291 tissue was significantly decreased in the CG group, PH group and B group (Fig. 4D). Similarly, HFD
292 feeding markedly enhanced cholesterol in the liver of rats and was further reversed to normal levels
293 by CG and PH treatment (Fig. 4E).

294

295 Fig 4. The effect of drugs on Hepatic inflammation and hepatic steatosis in HFD-fed rats. (A) Formalin-fixed and paraffin-embedded
296 liver of the C group, M group, CG group, PH group and B group were stained with hematoxylin & eosin (HE) and observed under an
297 optical microscope (HE, ×100, and ×400). Black arrows indicate steatosis of hepatocytes, red arrows indicate dotted necrosis areas.
298 (B) Representative oil red O staining of the liver. (C) The expression of lipid deposition of liver analyzed with ImageJ. (D, E) The level of
299 TG and cholesterol in the liver. Statistical analyses were conducted with one-way ANOVA. #P < 0.05, ##P<0.01, ###P < 0.001 versus C;
300 *P < 0.05, **P < 0.01, ***P < 0.001 versus model group.

301 3.5 Drugs inhibited the activation of nuclear factor κB signaling and decreased the content of
302 inflammatory cytokines (IL-1β, IL-6, TNF-α)

25 13
 26 Running Title

303 The expression levels of TLR4, AP-1, MyD88 and phosphorylated NF-κB p65 were increased
304 significantly in the liver tissue of the M group compared with that of the C group. After treatment,
305 the expression levels of TLR4, AP-1, MyD88, and phosphorylated NF-κB p65 in the liver tissue were
306 significantly decreased in the CG, PH, and B group (Fig. 5A-F). These results show that HFD-fed
307 rats produce higher levels of pro-inflammatory cytokines in hepatic and adipose tissue, including
308 tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL1β). The concentration of these
309 cytokines after 14 weeks of HFD feeding was measured. IL-1β, IL-6 and TNF-α content in the liver
310 tissue of the M group were significantly increased compared with that of the C group. After
311 treatment, the IL-1β, IL-6, TNF-α content in the liver tissue were significantly decreased in the CG,
312 PH and B group (Fig. 5G-I).

313

314 Fig 5. The effect of drugs on LPS-TLR4 signaling pathway activation in HFD-fed rats. (A) Representative western blots of TLR4, AP-1
315 and MyD88 protein expression in the liver are shown for each group. GAPDH was used as a loading control. (C-E) Bar graphs show
316 densitometry analysis of specific bands expressed as percentages relative to control. (B)A representative western blot of
317 phosphorylated NF-κB p65 in the liver of each group is shown. Total NF-κB p65 was used as a loading control, indicating its
318 phosphorylation level. (F) Bar graphs show densitometry analysis of specific bands expressed as percentages relative to the control.
319 (G-I) drugs decreased the content of inflammatory cytokines (IL-1β, IL-6, TNF-α) in the liver. The content of IL-1β, IL-6, and TNF-α in

27 14
 28 Running Title

320 each group. The LPS content in plasma. Statistical analyses were conducted with one-way ANOVA. #P < 0.05, ##P<0.01, ###P < 0.001
321 versus C; *P < 0.05, **P < 0.01, ***P < 0.001 versus model group.

322

323 Fig 6. Mechanistic diagram for this study.

324 4 Discussion

325 NASH is the inflammatory subtype NAFLD[38]. The mechanism by which NAFLD progresses to
326 NASH has not been fully elucidated. Despite only a few percent of NAFLD patients developing into
327 NASH and growing evidence from case series that non-obese individuals maybe also affected by
328 NASH. For a long time, weight loss through lifestyle modification was a common treatment for
329 NAFLD and NASH. However, our results shown drug treatments can effectively improve the NASH
330 by inhibiting LPS‐induced inflammation without weight loss.

331 Endotoxin/TLR4 signaling has been shown to be important in the activation of inflammatory
332 pathways linked to NASH in previous clinical trials.[39] LPS is the main component of endotoxin,
333 the primary constituent of the gram-negative bacterial ( GNB )outer membrane and cover
334 approximately 75% of the cell surface[40]. Increasing amount of evidence suggesting that gut-
335 derived LPS and the ensuing inflammatory response participated in the pathogenesis of NASH[41-
336 43]. The gut and the liver are anatomically linked, and intestinal barrier integrity prevents LPS from

29 15
 30 Running Title

337 being transported into the liver via the portal vein. [44]. LPS as blood marker have been used as
338 indicators of intestinal permeability,[45] and considered to be the major contributing factor to the
339 inflammatory cytokine storm caused by disruption of the gut barrier.[46]

340 Intestinal barrier repair is essential to further inhibits the LPS-induced inflammatory response.
341 Several studies have reported that HFD alter the composition of the gut microbiota which in turn
342 leads to an impaired gut barrier function.[47-49]. Recent studies have found HFD are associated with
343 a decrease in overall microbial abundance and diversity with a shift from Bacteroidetes to Firmicutes.
344 [50-52] The same phenomenon was observed in the present experiments, meanwhile drug treatments
345 restored bacterial diversity and significantly reduced F/B which is considered a hallmark of obesity.
346 (48, 49) Dysbiosis, or compositional changes in the intestinal microbiota, reduces the function of the
347 intestinal mucosal barrier and increases bacterial translocation, and intestinal pathogenic bacteria
348 destroy structural barriers by altering intestinal tight junction proteins.[53] Tight junction proteins are
349 important for the maintenance of intestinal barrier integrity[54], both ZO-1 and Occludin have been
350 shown to participate in tight junctions structural integrity by binding to a actin-cytoskeleton[55], can
351 enhance the homeostatic barrier function of primary cultured Sertoli cells[56]. The
352 immunohistochemical results of the small intestine also showed that drug treatments normalized the
353 expression of these two tight junction proteins which has been reduced by HFD. Together, these
354 results indicate that drug treatments can repair the intestinal barrier following an insult by
355 maintaining the balance of intestinal microflora and regulating tight junction protein. This was also
356 confirmed by the biochemical and histopathological outcomes. The most recent findings have linked
357 gut flora disruption and mucosal barrier deficiencies to higher inflammatory pathway activation.[55]
358 The disruption of the intestinal barrier function and the abnormality of the gut microbiota could be a
359 source of endotoxins. [57] drug treatments ameliorate the disruption of intestinal barrier function by
360 HFD and its resulting infiltration of LPS into the systemic circulation.

361 Disruption of barrier function causes allows the translocation of LPS into the liver via the portal vein
362 and promotes NASH development[58, 59]. After exposure to HFD, we report accompanied by
363 intestinal damage by intestinal damage several proinflammatory cytokines (IL-1β, IL-6, TNF-α) were
364 significantly increased. To this day, stimulation from gut-derived LPS may be most significant
365 predisposing factors for liver inflammation. However, we still lack a full understanding of the
366 inflammatory pathways induced by LPS in NASH. We investigated several key proteins involved in
367 the inflammatory pathways to further understand the mechanisms regulating this pathway. TLR4 is a
368 transmembrane protein of LPS pattern recognition receptors that regulates early inflammatory

31 16
 32 Running Title

369 responses and is vital in triggering the innate immune system. [60] Accumulating evidence indicates
370 that TLR4 is a starting point in LPS-induced inflammatory process[61] It is commonly held that
371 TLR4 promotes the production of inflammatory cytokines through two distinct signaling networks:
372 the MyD88-dependent and TRIF-dependent pathways[62] However, a recent study found mice
373 lacking MyD88 have been shown to be unable to produce TNF, IL-1β or IL-6 in response to LPS.
374 (17) This case illustrates that MyD88, but not TRIF, is an indispensable participant in LPS-induced
375 inflammatory process. MyD88-dependent rapid activation of the transcription factors AP-1 and NF-
376 ĸB which is typical in inflamed colonic tissue [63, 64], was promoted by TLR4 detection of LPS at
377 the plasma membrane. [16] The activation of the proinflammatory transcription factor AP-1,
378 neutrophil infiltration, and NF- B activation have all recently been identified as key contributors in
379 the development of NASH.[65] Additionally, there was also one study verified that MyD88
380 knockdown can reduce the activity of NF-κB and AP-1 pathways. [66] The AP-1 and NF-κB
381 inflammatory signal transduction pathways are the main links to the activation of the inflammatory
382 cascade.[67] And many studies have shown that by inhibiting AP-1 and NF-κB, the expression of
383 inflammatory cytokines can be reduced[62, 68-70]. At that point, it is tempting to speculate a
384 potential signalling pathway of gut-derived LPS- induced inflammatory process in NASH based on
385 previous studies. We subsequently confirmed this speculation experimentally. Furthermore, our
386 results also revealed that drug treatments ameliorate NASH by suppressing LPS- induced
387 inflammatory signaling pathways but not reducing fatty liver by body weight loss, thus suggesting
388 gut-derived LPS is the main stimulus of inflammatory responses in NASH.

389 Taken together, our work is the first in which we comprehensively studied the LPS-induced
390 inflammatory pathway activation in NASH, demonstrate that drug treatments successfully improved
391 NASH through stopping LPS enter liver and trigger an inflammatory response. (Fig. 6)
392

393 5 Conflict of Interest

394 The authors declare that the research was conducted in the absence of any commercial or financial
395 relationships that could be construed as a potential conflict of interest.

396 6 Author Contributions

397 Writing, ZZJ, XJ; Conceptualization, HQL. and LXX.; methodology, SLZ; software, SLX and ZSL;
398 validation, NQ, ZYG. and WYY; formal analysis, ZLM; investigation, YYM; resources, LPH.; data

33 17
 34 Running Title

399 curation, LXY; visualization, HSQ; supervision, CLF; project administration, LHQ; funding
400 acquisition, CSD. All authors have read and agreed to the published version of the manuscript.

401 7 Funding

402 This project was supported by the National Natural Science Foundation of China (No. 81873242,
403 82174141), National TCM Administration Young QI Huang Scholars Support Project ([2021] No.
404 200).

405 8 Acknowledgments

406 We wholeheartedly appreciate all members in Shaodong Chen’s research group for valuable
407 contributions and discussion to this project.

408

409 Reference

410

411 1. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ:
412 The diagnosis and management of non-alcoholic fatty liver disease: practice Guideline
413 by the American Association for the Study of Liver Diseases, American College of
414 Gastroenterology, and the American Gastroenterological Association. Hepatology 2012,
415 55(6):2005-2023.
416 2. Musso G, Cassader M, Gambino R: Non-alcoholic steatohepatitis: emerging molecular
417 targets and therapeutic strategies. Nat Rev Drug Discov 2016, 15(4):249-274.
418 3. Wesolowski SR, Kasmi KC, Jonscher KR, Friedman JE: Developmental origins of NAFLD:
419 a womb with a clue. Nat Rev Gastroenterol Hepatol 2017, 14(2):81-96.
420 4. Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres-Gonzalez A, Gra-Oramas B,
421 Gonzalez-Fabian L, Friedman SL, Diago M, Romero-Gomez M: Weight Loss Through
422 Lifestyle Modification Significantly Reduces Features of Nonalcoholic Steatohepatitis.
423 Gastroenterology 2015, 149(2):367-378 e365; quiz e314-365.
424 5. Arrese M, Cabrera D, Kalergis AM, Feldstein AE: Innate Immunity and Inflammation in
425 NAFLD/NASH. Dig Dis Sci 2016, 61(5):1294-1303.
426 6. Imani Fooladi A, Tavakoli H, Naderi A: Detection of enterotoxigenic Staphylococcus
427 aureus isolates in domestic dairy products. Iran J Microbiol 2010, 2(3):137-142.
428 7. Seki E, Schnabl B: Role of innate immunity and the microbiota in liver fibrosis: crosstalk
429 between the liver and gut. J Physiol-London 2012, 590(3):447-458.
430 8. Bryant CE, Symmons M, Gay NJ: Toll-like receptor signalling through macromolecular
431 protein complexes. Mol Immunol 2015, 63(2):162-165.

35 18
 36 Running Title

432 9. Anwar MA, Basith S, Choi S: Negative regulatory approaches to the attenuation of Toll-
433 like receptor signaling. Exp Mol Med 2013, 45:e11.
434 10. Roh YS, Seki E: Toll-like receptors in alcoholic liver disease, non-alcoholic
435 steatohepatitis and carcinogenesis. J Gastroenterol Hepatol 2013, 28 Suppl 1:38-42.
436 11. Rives C, Fougerat A, Ellero-Simatos S, Loiseau N, Guillou H, Gamet-Payrastre L, Wahli W:
437 Oxidative Stress in NAFLD: Role of Nutrients and Food Contaminants. Biomolecules
438 2020, 10(12).
439 12. Luther J, Garber JJ, Khalili H, Dave M, Bale SS, Jindal R, Motola DL, Luther S, Bohr S,
440 Jeoung SW et al: Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered
441 Intestinal Permeability. Cell Mol Gastroenterol Hepatol 2015, 1(2):222-232.
442 13. Grube BJ, Cochane CG, Ye RD, Green CE, McPhail ME, Ulevitch RJ, Tobias PS:
443 Lipopolysaccharide binding protein expression in primary human hepatocytes and
444 HepG2 hepatoma cells. J Biol Chem 1994, 269(11):8477-8482.
445 14. Poniachik J, Csendes A, Diaz JC, Rojas J, Burdiles P, Maluenda F, Smok G, Rodrigo R,
446 Videla LA: Increased production of IL-1alpha and TNF-alpha in lipopolysaccharide-
447 stimulated blood from obese patients with non-alcoholic fatty liver disease. Cytokine
448 2006, 33(5):252-257.
449 15. Sharma BR, Karki R, Lee E, Zhu Q, Gurung P, Kanneganti TD: Innate immune adaptor
450 MyD88 deficiency prevents skin inflammation in SHARPIN-deficient mice. Cell Death
451 Differ 2019, 26(4):741-750.
452 16. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S: Unresponsiveness of MyD88-deficient
453 mice to endotoxin. Immunity 1999, 11(1):115-122.
454 17. Ito D, Shimizu S, Inoue K, Saito D, Yanagisawa M, Inukai K, Akiyama Y, Morimoto Y,
455 Noda M, Shimada A: Comparison of Ipragliflozin and Pioglitazone Effects on
456 Nonalcoholic Fatty Liver Disease in Patients With Type 2 Diabetes: A Randomized, 24-
457 Week, Open-Label, Active-Controlled Trial. Diabetes Care 2017, 40(10):1364-1372.
458 18. Bril F, Biernacki DM, Kalavalapalli S, Lomonaco R, Subbarayan SK, Lai J, Tio F, Suman A,
459 Orsak BK, Hecht J et al: Role of Vitamin E for Nonalcoholic Steatohepatitis in Patients
460 With Type 2 Diabetes: A Randomized Controlled Trial. Diabetes Care 2019, 42(8):1481-
461 1488.
462 19. Jacob AN, Salinas K, Adams-Huet B, Raskin P: Weight gain in type 2 diabetes mellitus.
463 Diabetes Obes Metab 2007, 9(3):386-393.
464 20. Yu H, Liu L, Chang Z, Wang S, Wen B, Yin P, Liu D, Chen B, Zhang J: Genome Sequence
465 of the Bacterium Bifidobacterium longum Strain CMCC P0001, a Probiotic Strain Used
466 for Treating Gastrointestinal Disease. Genome Announc 2013, 1(5).
467 21. Yu HJ, Liu W, Chang Z, Shen H, He LJ, Wang SS, Liu L, Jiang YY, Xu GT, An MM et al:
468 Probiotic BIFICO cocktail ameliorates Helicobacter pylori induced gastritis. World J
469 Gastroenterol 2015, 21(21):6561-6571.
470 22. Wu J, Gan T, Zhang Y, Xia G, Deng S, Lv X, Zhang B, Lv B: The prophylactic effects of
471 BIFICO on the antibiotic-induced gut dysbiosis and gut microbiota. Gut Pathog 2020,
472 12:41.

37 19
 38 Running Title

473 23. Zhou Y, Zhang F, Mao L, Feng T, Wang K, Xu M, Lv B, Wang X: Bifico relieves irritable
474 bowel syndrome by regulating gut microbiota dysbiosis and inflammatory cytokines.
475 Eur J Nutr 2022.
476 24. Feng Q, Liu W, Baker SS, Li H, Chen C, Liu Q, Tang S, Guan L, Tsompana M, Kozielski R
477 et al: Multi-targeting therapeutic mechanisms of the Chinese herbal medicine QHD in
478 the treatment of non-alcoholic fatty liver disease. Oncotarget 2017, 8(17):27820-27838.
479 25. Feng Q, Gou XJ, Meng SX, Huang C, Zhang YQ, Tang YJ, Wang WJ, Xu L, Peng JH, Hu
480 YY: Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-
481 Activated Protein Kinase In Vivo and In Vitro. Evid Based Complement Alternat Med
482 2013, 2013:184358.
483 26. Feng Q, Cheng Y, Hu YY, Zhang H, Peng JH, Zhang N: Qushi Huayu Decoction () inhibits
484 protein and gene expression of cathepsin B in HepG2 cells induced by free fatty acids.
485 Chin J Integr Med 2010, 16(6):518-524.
486 27. Yin X, Peng J, Zhao L, Yu Y, Zhang X, Liu P, Feng Q, Hu Y, Pang X: Structural changes
487 of gut microbiota in a rat non-alcoholic fatty liver disease model treated with a Chinese
488 herbal formula. Syst Appl Microbiol 2013, 36(3):188-196.
489 28. Chen T, Liu S, Zheng M, Li Y, He L: The effect of geniposide on chronic unpredictable
490 mild stress-induced depressive mice through BTK/TLR4/NF-kappaB and BDNF/TrkB
491 signaling pathways. Phytother Res 2021, 35(2):932-945.
492 29. Zhang C, Wang N, Tan HY, Guo W, Chen F, Zhong Z, Man K, Tsao SW, Lao L, Feng Y:
493 Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF-1alpha-
494 independent VEGF expression and angiogenesis in hepatocellular carcinoma. Br J
495 Pharmacol 2020, 177(14):3240-3257.
496 30. Zhang H, Sun Y, Yau SY, Zhou Y, Song X, Zhang HT, Zhu B, Wu H, Chen G: Synergistic
497 effects of two naturally occurring iridoids in eliciting a rapid antidepressant action by
498 up-regulating hippocampal PACAP signalling. Br J Pharmacol 2022, 179(16):4078-4091.
499 31. Dusabimana T, Park EJ, Je J, Jeong K, Yun SP, Kim HJ, Kim H, Park SW: Geniposide
500 Improves Diabetic Nephropathy by Enhancing ULK1-Mediated Autophagy and
501 Reducing Oxidative Stress through AMPK Activation. Int J Mol Sci 2021, 22(4).
502 32. Wu CS, Chiang HM, Chen Y, Chen CY, Chen HF, Su WC, Wang WJ, Chou YC, Chang WC,
503 Wang SC et al: Prospects of Coffee Leaf against SARS-CoV-2 Infection. Int J Biol Sci
504 2022, 18(12):4677-4689.
505 33. Cai Y, Zhang Z, Jiang S, Yu M, Huang C, Qiu R, Zou Y, Zhang Q, Ou S, Zhou H et al:
506 Chlorogenic acid increased acrylamide formation through promotion of HMF
507 formation and 3-aminopropionamide deamination. J Hazard Mater 2014, 268:1-5.
508 34. Ke Y, Ma Z, Ye H, Guan X, Xiang Z, Xia Y, Shi Q: Chlorogenic Acid-Conjugated
509 Nanoparticles Suppression of Platelet Activation and Disruption to Tumor Vascular
510 Barriers for Enhancing Drug Penetration in Tumor. Adv Healthc Mater 2022:e2202205.
511 35. Bisht A, Dickens M, Rutherfurd-Markwick K, Thota R, Mutukumira AN, Singh H:
512 Chlorogenic Acid Potentiates the Anti-Inflammatory Activity of Curcumin in LPS-
513 Stimulated THP-1 Cells. Nutrients 2020, 12(9).

39 20
 40 Running Title

514 36. Wu X, Yang H, Chen X, Gao J, Duan Y, Wei D, Zhang J, Ge K, Liang XJ, Huang Y et al:
515 Nano-herb medicine and PDT induced synergistic immunotherapy for colon cancer
516 treatment. Biomaterials 2021, 269:120654.
517 37. Chen B, Zhang H, Qiu J, Wang S, Ouyang L, Qiao Y, Liu X: Mechanical Force Induced
518 Self-Assembly of Chinese Herbal Hydrogel with Synergistic Effects of Antibacterial
519 Activity and Immune Regulation for Wound Healing. Small 2022, 18(21):e2201766.
520 38. Noguchi R, Kaji K, Namisaki T, Moriya K, Kawaratani H, Kitade M, Takaya H, Aihara Y,
521 Douhara A, Asada K et al: Novel oral plasminogen activator inhibitor1 inhibitor TM5275
522 attenuates hepatic fibrosis under metabolic syndrome via suppression of activated
523 hepatic stellate cells in rats. Mol Med Rep 2020, 22(4):2948-2956.
524 39. Rivera CA, Adegboyega P, van Rooijen N, Tagalicud A, Allman M, Wallace M: Toll-like
525 receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-
526 alcoholic steatohepatitis. J Hepatol 2007, 47(4):571-579.
527 40. Nikaido H, Vaara M: Molecular basis of bacterial outer membrane permeability.
528 Microbiol Rev 1985, 49(1):1-32.
529 41. Mai W, Xu Y, Xu J, Zhao D, Ye L, Yu G, Wang Z, Lu Q, Lin J, Yang T et al: Berberine
530 Inhibits Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome
531 Activation and Pyroptosis in Nonalcoholic Steatohepatitis via the ROS/TXNIP Axis.
532 Front Pharmacol 2020, 11:185.
533 42. Lu Z, Li Y, Syn WK, Wang Z, Lopes-Virella MF, Lyons TJ, Huang Y: Amitriptyline
534 inhibits nonalcoholic steatohepatitis and atherosclerosis induced by high-fat diet and
535 LPS through modulation of sphingolipid metabolism. Am J Physiol Endocrinol Metab
536 2020, 318(2):E131-E144.
537 43. Lytle KA, Jump DB: Is Western Diet-Induced Nonalcoholic Steatohepatitis in Ldlr-/-
538 Mice Reversible? PLoS One 2016, 11(1):e0146942.
539 44. Liu L, Liu Z, Li H, Cao Z, Li W, Song Z, Li X, Lu A, Lu C, Liu Y: Naturally Occurring
540 TPE-CA Maintains Gut Microbiota and Bile Acids Homeostasis via FXR Signaling
541 Modulation of the Liver-Gut Axis. Front Pharmacol 2020, 11:12.
542 45. Derikx JP, Luyer MD, Heineman E, Buurman WA: Non-invasive markers of gut wall
543 integrity in health and disease. World J Gastroenterol 2010, 16(42):5272-5279.
544 46. Van Goor A, Ashwell CM, Persia ME, Rothschild MF, Schmidt CJ, Lamont SJ: Unique
545 genetic responses revealed in RNA-seq of the spleen of chickens stimulated with
546 lipopolysaccharide and short-term heat. PLoS One 2017, 12(2):e0171414.
547 47. Cani PD, Bibiloni R, Knauf C, Neyrinck AM, Neyrinck AM, Delzenne NM, Burcelin R:
548 Changes in gut microbiota control metabolic endotoxemia-induced inflammation in
549 high-fat diet-induced obesity and diabetes in mice. Diabetes 2008, 57(6):1470-1481.
550 48. de La Serre CB, Ellis CL, Lee J, Hartman AL, Rutledge JC, Raybould HE: Propensity to
551 high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and
552 gut inflammation. Am J Physiol Gastrointest Liver Physiol 2010, 299(2):G440-448.
553 49. Lam YY, Ha CW, Campbell CR, Mitchell AJ, Dinudom A, Oscarsson J, Cook DI, Hunt NH,
554 Caterson ID, Holmes AJ et al: Increased gut permeability and microbiota change
555 associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced
556 obese mice. PLoS One 2012, 7(3):e34233.

41 21
 42 Running Title

557 50. Hildebrandt MA, Hoffmann C, Sherrill-Mix SA, Keilbaugh SA, Hamady M, Chen YY,
558 Knight R, Ahima RS, Bushman F, Wu GD: High-fat diet determines the composition of
559 the murine gut microbiome independently of obesity. Gastroenterology 2009,
560 137(5):1716-1724 e1711-1712.
561 51. Zhang C, Zhang M, Pang X, Zhao Y, Wang L, Zhao L: Structural resilience of the gut
562 microbiota in adult mice under high-fat dietary perturbations. ISME J 2012, 6(10):1848-
563 1857.
564 52. Murphy EA, Velazquez KT, Herbert KM: Influence of high-fat diet on gut microbiota: a
565 driving force for chronic disease risk. Curr Opin Clin Nutr Metab Care 2015, 18(5):515-
566 520.
567 53. Ren X, Zhu Y, Gamallat Y, Ma S, Chiwala G, Meyiah A, Xin Y: E. coli O124 K72 alters the
568 intestinal barrier and the tight junctions proteins of guinea pig intestine. Biomed
569 Pharmacother 2017, 94:468-473.
570 54. Luo Y, Ma H, Niu S, Li X, Nie L, Li G: Effects of norepinephrine on colonic tight
571 junction protein expression during heat stress. Exp Ther Med 2021, 21(5):421.
572 55. Yang F, Wang A, Zeng X, Hou C, Liu H, Qiao S: Lactobacillus reuteri I5007 modulates
573 tight junction protein expression in IPEC-J2 cells with LPS stimulation and in newborn
574 piglets under normal conditions. BMC Microbiol 2015, 15:32.
575 56. Liu B, Shen LJ, Zhao TX, Sun M, Wang JK, Long CL, He DW, Lin T, Wu SD, Wei GH:
576 Automobile exhaust-derived PM2.5 induces blood-testis barrier damage through ROS-
577 MAPK-Nrf2 pathway in sertoli cells of rats. Ecotoxicol Environ Saf 2020, 189:110053.
578 57. Geurts L, Neyrinck AM, Delzenne NM, Knauf C, Cani PD: Gut microbiota controls
579 adipose tissue expansion, gut barrier and glucose metabolism: novel insights into
580 molecular targets and interventions using prebiotics. Benef Microbes 2014, 5(1):3-17.
581 58. Henao-Mejia J, Elinav E, Jin C, Hao L, Mehal WZ, Strowig T, Thaiss CA, Kau AL,
582 Eisenbarth SC, Jurczak MJ et al: Inflammasome-mediated dysbiosis regulates progression
583 of NAFLD and obesity. Nature 2012, 482(7384):179-185.
584 59. Zhu L, Baker SS, Gill C, Liu W, Alkhouri R, Baker RD, Gill SR: Characterization of gut
585 microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between
586 endogenous alcohol and NASH. Hepatology 2013, 57(2):601-609.
587 60. Lin WC, Deng JS, Huang SS, Wu SH, Chen CC, Lin WR, Lin HY, Huang GJ: Anti-
588 Inflammatory Activity of Sanghuangporus sanghuang Mycelium. Int J Mol Sci 2017,
589 18(2).
590 61. Wang S, Luo Q, Zhou Y, Fan P: CLG from Hemp Seed Inhibits LPS-Stimulated
591 Neuroinflammation in BV2 Microglia by Regulating NF-kappaB and Nrf-2 Pathways.
592 ACS Omega 2019, 4(15):16517-16523.
593 62. Yi W, Wen Y, Tan F, Liu X, Lan H, Ye H, Liu B: Impact of NF-kappaB pathway on the
594 apoptosis-inflammation-autophagy crosstalk in human degenerative nucleus pulposus
595 cells. Aging (Albany NY) 2019, 11(17):7294-7306.
596 63. Kim KA, Lee IA, Gu W, Hyam SR, Kim DH: beta-Sitosterol attenuates high-fat diet-
597 induced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide
598 to toll-like receptor 4 in the NF-kappaB pathway. Mol Nutr Food Res 2014, 58(5):963-
599 972.

43 22
 44 Running Title

600 64. Yan YX, Shao MJ, Qi Q, Xu YS, Yang XQ, Zhu FH, He SJ, He PL, Feng CL, Wu YW et al:
601 Artemisinin analogue SM934 ameliorates DSS-induced mouse ulcerative colitis via
602 suppressing neutrophils and macrophages. Acta Pharmacol Sin 2018, 39(10):1633-1644.
603 65. Liang W, Lindeman JH, Menke AL, Koonen DP, Morrison M, Havekes LM, van den Hoek
604 AM, Kleemann R: Metabolically induced liver inflammation leads to NASH and differs
605 from LPS- or IL-1beta-induced chronic inflammation. Lab Invest 2014, 94(5):491-502.
606 66. Zhu G, Cheng Z, Huang Y, Zheng W, Yang S, Lin C, Ye J: MyD88 mediates colorectal
607 cancer cell proliferation, migration and invasion via NFkappaB/AP1 signaling pathway.
608 Int J Mol Med 2020, 45(1):131-140.
609 67. Li J, Liu YH, Ou S, Dai XM, Wang JP, Su YP: Steroid receptor coactivator-3
610 differentially regulates the inflammatory response in peritoneal macrophages. Mol Med
611 Rep 2012, 5(4):1099-1105.
612 68. Yang WS, Jeong D, Nam G, Yi YS, Yoon DH, Kim TW, Park YC, Hwang H, Rhee MH,
613 Hong S et al: AP-1 pathway-targeted inhibition of inflammatory responses in LPS-
614 treated macrophages and EtOH/HCl-treated stomach by Archidendron clypearia
615 methanol extract. J Ethnopharmacol 2013, 146(2):637-644.
616 69. Wagner EF: Bone development and inflammatory disease is regulated by AP-1 (Fos/Jun).
617 Ann Rheum Dis 2010, 69 Suppl 1:i86-88.
618 70. Ren X, Wang L, Chen Z, Hou D, Xue Y, Diao X, Shen Q: Foxtail Millet Improves Blood
619 Glucose Metabolism in Diabetic Rats through PI3K/AKT and NF-kappaB Signaling
620 Pathways Mediated by Gut Microbiota. Nutrients 2021, 13(6).

621

45 23