international dental journal 7 4 ( 2 0 2 4 ) 1 1 9 − 1 2 8

Scientific Research Report

Identification the Low Oxidative Stress Subtype of

Periodontitis

Yuchen Wu a, Xianfang Zhang a, Yunong Chen a, Weiting Chen b,

Wenhao Qian c*
a
Department of Prosthodontics, Shanghai Xuhui District Dental Center, Shanghai, People’s Republic of China
b
Department of Periodontology, Shanghai Xuhui District Dental Center, Shanghai, People’s Republic of China
c
Department of Oral Implantology, Shanghai Xuhui District Dental Center, Shanghai, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Article history: Objective: The aim of this research was to identify the low oxidative stress−related genes
Received 25 April 2023 expression (L-OS) subtype in patients with periodontitis.
Received in revised form Methods: Microarray data (MA) were retrieved from the Gene Expression Omnibus database.
7 July 2023 The different oxidative stress (OS) subtypes of periodontitis were identified by K-means
Accepted 20 July 2023 clustering analysis and gene set variation analysis (GSVA). Differentially expressed genes
Available online 10 October 2023 (DEGs) (|Log fold change (FC)| >1, q < 0.05) amongst the OS subtypes and healthy controls
(HCs) were identified by Limma R package. The genomic feature of L-OS subtype and corre-
Key words: sponding medicines were evaluated and visualised with Drug-Gene Interaction Database
Periodontitis and cytoscape-v3.7.2 software (Pearson correlation coefficient > 0.4). Finally, the LASSO-
Low oxidative stress Logistic regression model was adopted to evaluate and predict patients’ OS phenotype in
Microarray data routine clinical practice.
TFs-Genes-Medicines network Results: The 241 periodontitis samples and 69 HCs were included. Thirty-three DEGs
Machine learning between the L-OS and high oxidative stress−related genes expression (H-OS) subtypes and
96 DEGs, including 8 transcription factors, between L-OS subtype and HCs were identified,
respectively. Then, the network of TFs-Genes-Drugs was constructed to determine geno-
mic feature of L-OS subtype. Finally, a 4-gene signature formula and the cutoff value were
identified by ML with LASSO model to predict patients’ classification.
Conclusions: For the first time, we identified L-OS subtype of periodontitis and evaluated its
genomic feature with MA.
Ó 2023 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction periodontitis is not fully understood, and no effective drugs

Periodontitis involves various chronic inflammations of tooth-
supporting structures including ligaments, gums, and bones.1,2
Periodontitis can cause tooth loss and exacerbate many medi-
cal conditions, such as atherosclerosis, type 2 diabetes mellitus,
and even cancer by increasing the inflammatory burden in the
periodontal microenvironment or throughout the body.3-5 Stud-
ies have shown that the incidence of periodontitis in the popu-
lation is about 30%, and more than one-third of them had
severe periodontitis, which confers major health and financial
burden to individuals and societies.6 To date, the aetiology of
* Corresponding author. No. 500 Fenglin Road, Xuhui District
Shanghai 200032, People’s Republic of China.
E-mail address: ORCID
Yuchen Wu: http://orcid.org/0000-0002-7596-9524
https://doi.org/10.1016/j.identj.2023.07.011
0020-6539/Ó 2023 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
have been approved for clinical treatment.7
Previous studies showed that the oxidative stress (OS) caused
by the imbalance of intracellular oxidant−antioxidant system
was entwined with periodontitis.8,9 When invaded by pathogenic
microorganism, the periodontal tissue releases pathogenic fac-
tors and recruited inflammatory cells, such as neutrophils,
monocytes, and macrophages. Phagocytes bring about bacteri-
cidal activity through “respiratory bursts” and released reactive
oxygen species (ROS).10,11 As the result of persistent chronic
inflammatory response, ROS produced excessively in the peri-
odontal microenvironment leading to OS, and eventual oxidative
damage to DNA, proteins, lipids. These may in turn lead to the
degeneration of periodontal ligament, fibrous connective tissue,
and alveolar bone.12 Hence reducing tissue OS damage should be
an effective method for managing periodontitis, but the out-
comes are variable and unsatisfactory, and the molecular
120 wu et al.
mechanisms underlying this phenomenon are not clear.13 To
date, whether the expression levels of OS-related genes are dis-
crepant in periodontitis or critical for routine clinical practice is
still unknown, and it is challenging to obtain reliable and quanti-
tative OS measurement values from a considerable number of
human samples. Further efforts are therefore needed to explore
the pathogenesis of periodontal disease and develop novel thera-
peutic drugs.
Recent advances in high-throughput sequencing and data
analysis have made it possible to monitor, quantify, and corre-
late biological signals with cellular phenotypes.14,15 As one of
the transcriptomics, microarray gene expression analysis can
detect differential genes expression for a multitude of RNAs
simultaneously, which makes it feasible to assess the activation
of OS-related pathways comprehensively.16 Data-driven
machine learning (ML) has gained increased attention from
researchers due to its superior performance in transforming
biological detection and treatment of human diseases.17 Vari-
ous clustering methods have been used in combination with
ML, such as K-means and Pam cluster analysis. The application
of combination high-throughput phenotypic screening and ML
could contribute to deep understanding the pathogenesis of
periodontitis.
Thus, the aim of the current study is to assess the expres-
sion levels of OS-related genes in patients with periodontitis
and identify the low oxidative stress−related genes expres-
sion (L-OS) subtype as well as the related genomic features by
using microarray data and ML.
Methods
Data acquisition and processing
In all, 241 samples with periodontitis and 69 control samples
were included in GSE16134, which was downloaded from the
Gene Expression Omnibus (GEO) database of National Center
for Biotechnology Information (NCBI)18,19 (http://www.ncbi.
nlm.nih.gov/geo/). The inclusion criteria for all participants
were as follows: at least 13 years old, not pregnant, not using
tobacco products or nicotine substitutes, have more than 24
teeth, and have not been on systemic antibiotics or anti-
inflammatory medications for more than 6 months. The
patients with periodontitis also need to meet the following
criteria: bleeding on probing, probing pocket depth ≥4 mm,
and clinical attachment loss ≥3 mm.18,19 The healthy and
periodontal pathology gingival tissue biopsies were obtained
from healthy controls and patients with periodontitis.18,19
Microarray data were centralised by log2 (x+1) transformation
and normalised by using the “normalize between array” func-
tion of R package “limma.”20 Meanwhile, 16 OS pathways and
the 580 OS-related genes were obtained from the Gene Ontol-
ogy (GO) resource (http://geneontology.org/).
Identification of the L-OS subtype of periodontitis
As an unsupervised learning technique, K-means clustering is a
commonly used clustering method. The value of 580 OS-related
genes were applied as the input feature of K-means clustering
to identify the L-OS of the 241 samples with periodontitis,
which was completed by R package ConsensusClusterPlus.21
Pam cluster algorithm, which is more robust than the K-m
method and effective for a small dataset due to its insensitivity
to outliers, was adopted here. GSVA, a method for the unsuper-
vised classification of samples, was adopted to evaluate the
expression of OS gene sets between subgroups.
Identification of the genomic features for the L-OS subtype of
periodontitis
To identify the genomic feature of the L-OS subtype of peri-
odontitis, differential analysis was applied between Cluster 1,
lowly enriched in OS pathways, and normal periodontal tis-
sues. Limma R package (version 4.0.4.), a commonly used differ-
entially expressed genes (DEGs) identification method, was
adopted here, and genes with Log fold change (FC) >1 or < 1,
and adjusted P value< .05 were deemed to be DEGs. Subse-
quently, those DEGs were divided into transcription factors
(TFs) and their targeting genes by the Pearson correlation anal-
ysis (PCA). Significant TFs−genes interaction pairs were
selected if their Pearson correlation coefficient (PCC) was >0.4.
Next, by integrating data from the Drug-Gene Interaction Data-
base (DGIdb), a public database providing information of inter-
action between drugs and genes (https://dgidb.genome.wustl.
edu/), the potential medicines for the key TFs and genes in the
L-OS subtype were also located. At last, the TFs-Genes-Medi-
cines network was visualised by cytoscape-v3.7.2 software22 to
guide drug application in the clinical practice.
ML-based 4 genes signature to recognise the L-OS subtype of
patients with periodontitis in clinical practice
To guide clinical classification, we further constructed an ML-
based 4-gene signature to commodiously distinguish patients
into L-OS and high oxidative stress−related genes expression
(H-OS) group, and subsets of marker genes and OS score were
chosen by using the Lasso regression algorithm. First, differen-
tial analysis was carried out between Cluster 1 and Cluster 3,
which are lowly and highly enriched in OS pathways, respec-
tively. Then, the DEGs between the 2 clusters were subjected to
logistic regression model with LASSO penalisation. Accordingly,
the OS score was computed for each patient by the following
formula: Sni Coefi*Xi (Coefi: logistic regression coefficient, Xi:
expression value of corresponding gene). Then, 70% of periodon-
titis samples were assigned to the training set to generate the 4-
gene signature formula by using the ML, and the remaining 30%
of periodontitis samples were assigned as the test set to test the
performance of this model. Finally, receiver operating character-
istic (ROC) curve and area under the curve (AUC) of ROC were
taken into consideration to evaluate the model’s performance in
both training and validation sets.
Statistics
Data processing and all analyses were accomplished by R 4.0.4.
(Package: limma, ggplot2, GSVA, glmnet, ConsensusClusterPlus,
Goplot, and so on). Chi-square test was used for counting data.
Wilcox or Kruskal−Wallis test were applied for comparisons
between groups, whilst Pearson and Spearman rank correlation
were adopted to estimate the statistical correlation of
 i d e n t i fi c a t i o n t h e l o w o x i d a t i v e s t r e s s s u b t y p e o f p e r i o d o n t i t i s
parametric or nonparametric variables. Two-sided P < .05 was
considered as significant threshold for all statistical tests.
This study was based on secondary database which is pub-
licly available in GEO (https://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi?acc=GSE16134).
Results
Identification of the L-OS subtype of periodontitis
The workflow is depicted in Figure 1. The baseline features of
samples collected from the GEO database are described in Sup-
plementary Table 1. First, microarray data of GSE1613418,19
were retrieved and included. In all, 580 genes were extracted
from 16 OS Go items, subjected to cluster analysis. Four optimal
clusters were obtained from Pam cluster analysis (Figure 2A)
according to the results of consensus of Cumulative Distribu-
tion Function (CDF) and area under CDF (Figure 2 B and C). One
Fig. 1 – The flow chart of this study.
121
cluster was excluded due to its small sample size (only 3 sam-
ples for each analysis) (Figure 2D). These 3 remaining clusters
demonstrated huge differences in GSVA pathway enrichment
analysis (Supplementary Table 2). Cluster 1 (67 of 241 samples)
was seen to be lowly enriched in oxidative-related pathways,
whilst Cluster 3 (98 of 241 samples) displayed higher enrich-
ment scores in the same genesets (Figure 2E). Accordingly,
about one-quarter of samples in Cluster 1 seem to be the L-OS
subtype of periodontal disease, which is less likely to respond
to antioxidant than Cluster 3 (Figure 2E).
The genomic features of the L-OS subtype of periodontial
disease
We identified L-OS subtype genomic signatures by comparing
periodontitis tissues with healthy controls (HCs). As the
results show in Figure 3A, 96 DEGs were identified between
Cluster 1 and HCs, and the list of the DEGs is described in Sup-
plementary Table 3. Amongst them, 53 genes were up-
122 wu et al.

Fig. 2 – Identification of the low oxidative stress−related genes expression subtype of periodontitis. A, Pam-clustering analysis of
all samples according to their enrichment degree of oxidative stress (OS) genes. B and C, The consensus Cumulative Distribution
Function (CDF) and delta area of the CDF curve. D, The tracking plot of CDF. E, GSVA pathway enrichment analysis.

regulated and 43 genes were down-regulated (Figure 3B) in
Cluster 1. The functional enrichment analysis showed the
most significant different Kyoto Encyclopedia of Genes and
Genomes (KEGG) item were seen to be mainly enriched in
“cell adhesion,” “leukocyte migration,” “macrophage migra-
tion pathways,” and so on (Figure 3C), and the related genes
are illustrated in Figure 3D. As leukocytes have been given
a key role in the process of oxidation in periodontal dis-
ease, these results also provided an indirect evidence for
Cluster 1 to be the L-OS subtype. The GO analysis revealed
that the prominent biological processes “extracellular
matrix organisation” and “extracellular structure organ-
isation” were significantly different between these 2
groups (Figure 3E).
Amongst this 96 DEGs, 8 TFs (ZBTB43, TET2, BNC1, CEBPG,
GRHL3, ISL1, FLI1, and ZNF57) came into our view for their
close connection with other genes in PCA (Figure 4A). Espe-
cially, TET2, BNC1, and FLI1 seemed to be in the dominant
position, for they had dozens of connections with other
genes. Then, a network of TFs-Genes-Drugs was constructed
by integrating drug−gene interaction information from DGIdb
(Figure 4B).
ML-based 4-gene signature to recognise the L-OS subtype of
periodontal disease in clinical practice
We applied an ML algorithm to develop a predictive diagnos-
tic model using periodontitis samples. First, 33 DEGs between
 i d e n t i fi c a t i o n t h e l o w o x i d a t i v e s t r e s s s u b t y p e o f p e r i o d o n t i t i s
Fig. 2 Continued.
the L-OS and H-OS subtypes (Cluster 1 and Cluster 3) were
identified by limma and admitted as the input features
(Figure 5A). The least absolute shrinkage and selection opera-
tor (LASSO)-Logistic regression model was adopted to predict
patients’ OS phenotype (Cluster 1 or Cluster 3), with 70% of
samples being the training set and the remaining 30% as the
test set. Then, the 4-gene (AADACL2, FCRL5, SLAMF7, and
COMP) signature formula was constructed and applied to pre-
dict patients’ classification by LASSO model, and the coeffi-
cient for each gene is described in Supplementary Table 4.
ROC was applied to evaluate the performance of the LASSO
logistic regression model in both datasets. The obtained diag-
nostic prediction model discriminated patients with L-OS
from periodontitis samples with high accuracy, and the AUC
was 0.902 for the training set and 0.863 for the validation set
(Figure 5B). In addition, the optimal threshold value of OS
123
score was calculated to help guide clinical classification, and
1.41 was the best cutoff value to stratify patients into L-OS
and H-OS subtypes (Figure 5C and D).
Discussion
Periodontitis is the sixth most prevalent disease in humans,
and as a chronic common disease amongst the elderly popula-
tion, the incidence of periodontitis can reach to 70% in people
older than 65 years, which brings an escalating burden to the
health care economy, especially with the ageing of the global
population.23 Periodontitis has irreversible adverse effects on
tooth-supporting structures and can cause tooth loss, poor
nutritional status, and reduced quality of life.1,2 Studies have
proved that severe periodontitis is a potential risk factor for
124 wu et al.

Fig. 3 – The differentially expressed genes (DEGs) and functional enrichment analysis between low oxidative stress−related
genes expression (L-OS) and control group. A, The heatmap and volcano plot (B) of DEGs between L-OS and control group. The
functional enrichment analysis showed the most significant different Kyoto Encyclopedia of Genes and Genomes (KEGG)
item and related genes (C, D) and most significant Gene Ontology (GO) items (E).

many systematic diseases, such as hypertension, atherosclero- tooth scaling, periodontal pocket flush, and guided tissue
sis, type 2 diabetes mellitus, and cancer.3,4 As a complex group regeneration) are variable and unsatisfactory.13
of inflammatory diseases, the aetiology of periodontitis is not In this study, we identified L-OS subtype of periodontitis and
fully understood, and the outcomes of clinical procedures (eg, evaluated its genomic feature by using bioinformatics
 i d e n t i fi c a t i o n t h e l o w o x i d a t i v e s t r e s s s u b t y p e o f p e r i o d o n t i t i s 125

Fig. 4 – The network of TFs-Genes-Drugs. The heatmap of 8 transcription factors (TFs) (A), and the network was constructed
by integrating drug−gene interaction information from DGIdb, and the genes with red background were key differentially
expressed genes (DEGs), the yellow background were TFs, and the compounds marked with blue background were drugs for
low oxidative stress−related genes expression (L-OS) (B).

algorithm with microarray data in a pioneering way. We then
identified a 4-gene signature formulae and the cutoff values to
identify this subtype in clinical practice using ML. Our data may
provide a theoretical justification for use of antioxidants for
periodontitis. Due to the short survival time, ROS are difficult to
detect. In previous studies, the level of OS in periodontitis was
analysed using indirect OS markers, such as the tissue expres-
sion levels of superoxide dismutase and catalase.24 In our study,
we took an innovative approach to examine the expression lev-
els of all OS-related genes with high-throughput microarray
data and successfully categorised the expression levels of OS-
related genes into 2 distinct subgroups: the L-OS and H-OS sub-
group. To the best of our knowledge, this is the first study to
report the L-OS subtype of periodontitis.
Many studies have proved that periodontitis is initiated by
uncontrolled inflammatory response, and elevated OS plays a
vital role in periodontitis by increasing periodontal microenvi-
ronmental inflammation.12,25 That means that conventional
treatment for periodontitis that focus on combating bacterial
pathogens appear to be insufficient, and providing additional
antioxidants, such as resveratrol—which react with ROS stoi-
chiometrically—has emerged as a promising treatment for
periodontitis.9,26 But recently, studies have shown that the vari-
able therapeutic effect of antioxidants in patients with peri-
odontitis.27-29 Evaluating the expression levels of OS-related
genes for clinicians before using antioxidants in periodontal
treatment might therefore be clinically relevant.
KEGG pathway enrichment analysis of 96 DEGs in L-OS vs
controls suggested that the signaling pathways were essen-
tially enriched in cell adhesion, leukocyte migration and mac-
rophage migration pathways. It has been confirmed that cell
adhesion molecules (such as vascular cell adhesion molecule
1) can promote arterial inflammation by trapping inflamma-
tory cells such as macrophages and T lymphocytes at sites of
inflammation.30 Since the inflammatory environment
strongly enhances phagocyte migration and activities, leuko-
cyte migration and macrophage migration have been given a
key role in the process of oxidation in periodontal disease,
126 wu et al.

Fig. 5 – The identification of low oxidative stress−related genes expression (L-OS) subtype of periodontal disease in clinical
practice. A, The volcano plot of differentially expressed genes (DEGs) between L-OS and high oxidative stress−related genes
expression (H-OS). B, The AUC of 4-genes-signature to predict patients’ classification. C,The performance identification of
LASSO model in training group and validation group. AUC, area under the curve.

and these results provided indirect evidence for Cluster 1 to
be L-OS subtypes.31,32 GO analysis revealed that the promi-
nent biological processes “extracellular matrix organisation”
and “extracellular structural organisation,” which were
related to tissue fibrosis atrophy, were also upregulated in
periodontitis.33,34 Amongst the 8 TFs predicted in the present
study, TET2, BNC1, and FLI1 have been verified as key modu-
lators and potential therapeutic targets in a wide variety of
inflammatory diseases.35-37
Recently, several automated ML strategies have been
developed to excavate potential prognostic biomarkers.38
Pam cluster analysis is a multivariate statistical analysis
method that classifies samples or indicators without prior
knowledge. The LASSO model regression algorithm can real-
ise the selection of independent variables and estimates
model parameters at the same time by using L1-regularised
linear regression.39 In this study, we integrated microarray
data and ML algorithm to identify the L-OS subtype of
 i d e n t i fi c a t i o n t h e l o w o x i d a t i v e s t r e s s s u b t y p e o f p e r i o d o n t i t i s
periodontitis and its genomic feature, as well as the diagnos-
tic criteria. First, we used OS pathway genes as a reference to
detect the validity of its subsets in the range of 2 to 10 and
combined with CDF and delta area of CDF curve to determine
the optimal number of clusters through Pam cluster analysis.
When the optimal number of subsets is 4, the area under CDF
no longer changes significantly (Figure 2B and C). For each
event, the leftmost clustering, containing a very small num-
ber of samples, was stable amongst analyses (Figure 2D),
which is estimated to be outliers. After being excluded from
the leftmost clustering, the distribution of the remaining 3
types of samples was found to be more uniform. Further-
more, 16 OS gene sets enrichment scored were evaluated by
GSVA in 3 remaining clusters, and L-OS and H-OS units were
determined. Then, the clinical diagnosis model was estab-
lished by LASSO. We identified a 4-gene diagnostic prediction
models that can specifically distinguish the L-OS whilst the
value of OS score is <1.4. This diagnostic model provides a
reference for clinical treatment of antioxidant stress in
patients with periodontitis.
There are some limitations to this study. Given the inher-
ent issues related to bioinformatics analysis— the conclu-
sions of this study may be constrained. The levels of
traditional OS indicators, superoxide dismutase, total antioxi-
dant capacity, and malondialdehyde, for example, also need
evauation to test the OS state of periodontal tissue in the
L-OS group. Further multicentric clinical studies are war-
ranted to validate our results under varying circumstances
and confirm whether these subtypes have different clinic
prognosis as well as their response to antioxidant therapy.
Conclusions
For the first time, we identified L-OS subtype of periodontitis
and evaluated its genomic features with microarray data. We
first identified a 4-gene signature formula and its cutoff value
to identify this subtype in clinical practice by ML algoruthms.
Our findings may provide a theoretical justification for the
use of antioxidants to manage peridontal disease.
Conflict of interest
None disclosed.
Acknowledgements
We acknowledge the GEO database for providing their plat-
forms and Papapanou et al. for uploading their meaningful
datasets.
Author contributions
All authors contributed to various parts of this manuscript.
Yuchen Wu was the major contributor in writing the manu-
script. Xianfang Zhang, Yunong Chen, and Weiting Chen helped
perform the bioinformatics analysis and machine learning.
127
Wenhao Qian designed this study and revised articles. All
authors have read and approved the final manuscript.
Funding
This study was funded by the National Natural Science Foun-
dation of China (no. 52272283) and Xuhui District Medical
Research Project of Shanghai (no. SHXH202141).
Supplementary materials
Supplementary material associated with this article can be
found in the online version at doi:10.1016/j.identj.2023.07.011.
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