Therapeutic effects of Zuojin Pill on Helicobacter pylori-induced chronic atrophic gastritis through JMJD2B/COX-2/VEGF signaling axis

Background: Zuojin Pill (ZJP), a famous Chinese medicinal formula, widely accepted for treatment of chronic atrophic gastritis (CAG) in China. This study aimed to explore the therapeutic effects and mechanisms of ZJP in Helicobacter pylori (H. pylori) - induced chronic atrophic gastritis (CAG) in vivo and in vitro. Methods: CAG rat model was induced by H. pylori. ZJP (0.63, 1.26, and 2.52 g/kg, respectively) was administered orally for four weeks. Therapeutic effects of ZJP were identi�ed by H&E staining and serum indices. In addition, cell viability, morphology and proliferation were detected by cell counting kit-8 (CCK8) and high-content screening assay (HCS), respectively. Moreover, relative mRNA expression and protein expression related to JMJD2B/COX-2/VEGF axis was detected to investigate the potential mechanisms of ZJP in CAG. Results: Results showed the symptoms (weight loss and gastric mucosa damage) of CAG were alleviated, and the contents of TNF-α in serum was markedly decreased after treating with ZJP. Moreover, cell viability, proliferation and morphology changes of GES-1 cells were ameliorated by ZJP intervention. In addition, proin�ammatory genes and JMJD2B/COX-2/VEGF axis related genes were suppressed by ZJP administration in vitro and in vivo. Meanwhile, immunohistochemistry (IHC) and western blot con�rmed down-regulation of these genes by ZJP intervention. Conclusion: ZJP treatment can alleviate gastric mucosal damage induced by H. pylori via JMJD2B/COX-2/VEGF axis.


Background
Chronic atrophic gastritis (CAG) is a universal disease in digestive system and one of the most continuous health concerns worldwide.Helicobacter pylori (H.pylori) infection is the strongest risk factor for gastric carcinogenesis [1].H. pylori infection can induce CAG, which develops through the premalignant periods of intestinal metaplasia, and dysplasia, before eventually leading to gastric cancer [2].At present, there are traditional options against H. pylori, including conventional triple therapy, bismuth based quadruple therapy and proton pump inhibitors [3].Whether it is triple therapy, quadruple therapy or proton pump inhibitors, conventional therapies can cause a series of serious adverse reactions, such as abdominal pain, constipation, decline of eradication rates.Therefore, novel and safe drugs are badly needed to be discovered and used in clinical treatment as soon as possible.Copious evidences from China declare traditional Chinese medicine (TCM) possess unlimited potential in treating H. pylori induced CAG.As an alternative therapy, TCM are gaining increasing popularity worldwide for the clinical treatment [4].Zuojin pill, recorded in the Danxi's experiential therapy, has been used for the treatment of gastrointestinal diseases for more than 700 years.However, the mechanism underlying the effect of ZJP in the treatment of H. pylori-induced CAG remains unclear.
ZJP contains Coptidis Rhizoma (CR) and Euodiae Fructus (EF) in the ratio of 6: 1 (w/w), which was initially recorded in an ancient medicine treatise, during China's Yuan Dynasty for treating gastro-intestinal disorders.CR is the dried rhizome of Coptis chinensis Franch., Coptisdeltoidea C.Y. Cheng et Hsiao, or Coptis teeta Wall.(Chinese Pharmacopoeia Commission 2015).In clinical, it is often utilized to treat diarrhea, abdominal fullness, vomiting, jaundice, toothache, high fever coma, diabetes and eczema [5].EF is the dried and immature fruit of Euodia rutaecarpa (Juss.)Benth., Euodia rutaecarpa ( Juss. ) Benth.var.o cinalis (Dode) Huang or Euodia rutaecarpa (Juss.)Benth.var.bodinieri (Dode) Huang (Chinese Pharmacopoeia Commission 2015).EF is widely applied for the treatment of in ammation, headache, and hypertension [6].Alkaloids are proved to be the primary compounds of CR and EF, including berberine, coptisine, palmatine, evodiamine and rutaecarpine.ZJP was o cially listed in the Chinese Pharmacopoeia (2015 edition) as a common prescription employed in clinical patients, who suffer from esophagitis, gastritis, peptic ulcer, and other disorders.In traditional Chinese formula, ZJP is often used for the treatment of CAG [7].In addition, ZJP and its active ingredients have obvious therapeutic effects against both in ammation and gastric mucosal damage in the stomach [8][9].Recent study has exhibited the gastrointestinal regulating functions of ZJP by restoring gastric electrical rhythm [10].Up to now, ZJP has been well-practiced in clinical application.The mechanism of ZJP acting on CAG is still unclear.In this study, we aimed to elucidate the effects and the molecular mechanisms of ZJP in H. pylori-induced CAG.
It has been reported that histone demethylase JMJD2B in stomach tissues when H. pylori infection became increased expression, and Cyclooxygenase-2 (COX-2) upregulated as downstream target protein of JMJD2B in H. pylori induced in ammatory process [11].Histone modi cation is an epigenetic mechanism, which plays a crucial role in gastric cancer carcinogenesis [12].JMJD2B was newly con rmed and characterized as a member of the histone demethylase JMJD2 family.Overexpress of JMJD2B is in gastric cancer can accelerate cell proliferation, survival, invasion and metastasis of gastric tumor [13].H. pylori infection activates the JMJD2B promoter and upregulates expression at transcriptional level.Recently, evidence has shown that that JMJD2B is required for H. pylori-induced COX-2 activation.COX-2 is involved in in ammation as a key enzyme in the synthesis of prostaglandin and overexpresses after H. pylori infection [14].The expression level of COX-2 is low under resting conditions in most cells, but can be induced by H. pylori in a cag T4SS-dependent manner [15].In addition, JMJD2B was shown to regulate vascular endothelial growth factor (VEGF), phosphoinositide 3kinase pathways, and angiogenesis, all of which play important roles in in ammation or tumorigenesis [16].It is well known that in H. pylori-infected gastritis, the concentration of angiogenic factor increases, resulting in the formation of new blood vessels.New angiogenesis will enhance supplement of nutrient and oxygen, and thus promote the development of gastritis.COX-2 is the key target responsible for promoting angiogenesis, which stimulate Vascular endothelial growth factor (VEGF) expression induced by H. pylori [17].
In this study, we explored the curative effect of ZJP in H. pylori induced CAG in vivo and in vitro.Moreover, we attempted to conduct a preliminary examination of the roles of JMJD2B/COX-2/VEGF axis in mechanism of ZJP for better understanding protective effects of ZJP in CAG.

Preparation of ZJP
Coptidis Rhizoma and Euodiae Fructus were soaked in pure water (6/1, w/w) for 30 min and were extracted twice (1 hour each time).Then, the extract was collected and evaporated to prepare dried powder under reduced pressure, respectively.Finally, the weight ratio of ZJP was 25.59%.ZJP powder was kept at 4℃ until oral administration to rats.ZJP was dissolved in and acted on GES-1 cells.ZJP powder was dissolved in dimethyl sulfoxide (DMSO) to con gure as mother liquor and then Dulbecco's modi ed Eagle's medium (DMEM) was used to dilute to corresponding concentration for using.
Bacterial strain and culture condition H.pylori isolated strain (ICDC111001) was kindly provided by Dr. Jianzhong Zhang (Chinese Disease Control and Prevention Center, Beijing, China).H. pylori strain was maintained and grown on Columbia blood agar (Thermo Fisher Scienti c, China), with incubation under micro-aerobic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ) at 37°C.After three to ve days' culture, bacteria strain was collected and adjusted to 1.0×10 8 colony forming units (CFU) /mL.

Animal experiments
Thirty-six male Sprague-Dawley (SD) rats were raised normally until Serum Tumor Necrosis Factor -α (TNF-α) and VEGF measurements The serum levels of TNF-α and VEGF were measured on a Synergy H1 Hybrid Reader (Biotech, USA).The measurement steps were conducted as per the manual of the ELISA kit (MLBIO biotechnology Co., Ltd., Shanghai, China).

Cell viability assay and H. pylori infection
The GES-1 cells were obtained from the FuHeng Cell Center, (Shanghai, China), which were cultivated in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a constant incubator containing 5% CO 2 at 37°C.The GES-1 cells were cultured overnight to reach at least 80% con uency.Cell viability was detected by cell counting kit-8 (CCK-8; Lot.PG658, DOJINDO, Japan).The optical density (OD) value was measured at 450 nm by using a Synergy H1 Hybrid Reader (Biotech, USA).
The H. pylori strain was harvested from Columbia blood agar plates, suspended in antibiotic-free DMEM medium complemented with 10% FBS, and then was added to the GES-1 cells culture.The H. pylori added to GES-1 cells at a multiplicity of infection (MOI) ratio of 10:1, 20:1, 50:1 and 100:1 for 0, 6, 12 and 24 h.
Bacterial counting of H. pylori was examined through Synergy H1 Hybrid Reader (Biotech, USA).The measurement of OD value was set at 600 nm to count colony forming units of H. pylori (1 OD 600nm =1.5×10 8 CFU/ml).Cocultivation was maintained at 37°C in a 5% CO 2 atmosphere.

High-Content Analysis Experiments (HCS)
Nuclear, cell morphology and the number of dead cells and living cells were detected by Array Scan High-Content System (Thermo Scienti c, Massachusetts, USA) [18].Hoechst 33342 (H3570, Invitrogen), calcein AM (C3099, Invitrogen), and ethidium homodimer-1 (EthD-1) (L3224, Invitrogen) were applied to quantify the GES-1 cells.Cell health pro ling assay module was selected in the HCS system, and several different wavelength channels were set to collect uorescence images.The measured parameters and format were similar to those used previously [19].Array Scan XTI (The Array Scan software algorithm was used to perform analysis) was used to quantify the mean uorescence intensity of GES-1 cells.

Real-time quantitative PCR Analysis in Vivo and in Vitro
Total mRNA of all rats' gastric tissue and GES-1 cells were extracted by TRIzol reagent (Nordic Bioscience, Beijing, China) and transformed into cDNA by reverse transcription kit (Promega, Madison, USA) according to the instructions.RT-qPCR for mRNA of JMJD2B, COX-2, VEGFR1, VEGFR2 and VEGF in rats and GES-1 cells were performed using SYBR Green PCR Master Mix (Nordic Bioscience, Beijing, China).Primer sequences are listed in Table 1.RT-qPCR was conducted on the 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA).Results were shown and exported in 7500 software (Applied Biosystems for 7500 and 7500 Fast Real-Time PCR Products, version 2.0.5).The relative amounts of mRNA were determined based on 2 −∆∆Ct calculations with β-actin as the endogenous reference.

Statistical Analysis
All results were presented as mean ± standard deviation (SD) and analyzed with the SPSS software program (version 19.0; SPSS Inc., Chicago, IL, USA).The differences were considered to be statistically signi cant when P < 0.05 and highly signi cant when P < 0.01.

Macroscopic pathology
Firstly, after 8 weeks, the rapid urease test in the model group was positive (Fig. 1A).After 4-week ZJP administration, the result of rapid urease test in low dose group was negative (Fig. 1B).During 8-week H. pylori infection, the rats induced by H. pylori exhibited CAG symptoms, such as weight loss, diarrhea and loss of appetite (Fig. 1C).The gastric mucosa of rats in model group showed paleness and thinning of gastric mucosa, with disarrayed plicae and small white nodules (Fig. 1D).

Histological examination of gastric mucosa
Histological features were critical evidence for the therapeutic effects of ZJP against H. pylori-induced CAG.Rats in the control group showed mucosal intact with tightly, abundant and orderly gastric glands.
CAG model rats showed inherent glands was missing, part of the mucosa was stripped, and neutrophils were in ltrated in the mucosa.Conversely, pathological changes in the omeprazole and the ZJP groups showed signi cantly improved in terms of the degree of edema, hyperemia, erosion and atrophy of gastric mucosa.Administration of high dose ZJP particularly exhibited lower in ammatory cell in ltration and gastric mucosal injury.Results were presented in Fig. 1E.

Cell viability
CCK8 was used for determination of the optimal concentration for ZJP administration.(Fig. 2A).The results showed that ZJP treatment for 24 h potently suppressed cell viability in a concentrationdependent manner with the increasing concentration (0, 10, 20, 30, 40, 60 and 120 μg/ml).The results showed that 120 μg/ml of ZJP could signi cantly inhibit the cell viability (P < 0.01).When 60 μg/mL of ZJP was given, the cell viability was close to 100%.Accordingly, 60 μg/mL of ZJP played a relatively protective role and was used as the optimal concentration to investigate the protective effects.For the in vitro study, 30 and 60 μg/mL of ZJP were used as low-dose and high-dose to GES-1 cells, respectively.

High-Content Analysis Experiments
In order to further determine the in uence of ZJP on cell morphology, HCS was used to investigate the effect of ZJP on the morphology of GES-1 cells.Nucleus staining (blue uorescence), cell cytoplasm labeling (green uorescence), and dead cells (red uorescence) were marked by Hoechst 33342, calcein AM, and EthD-1, respectively (Fig. 3A).In the control group, nucleus and cytoplasm of GES-1 cells possessed a homogenous Hoechst and calcein AM uorescence.After infection of H. pylori, there were cell viability, cell number and morphological changes, such as nuclear deformations, cell count decreased, green and red uorescence reduced and increased, respectively.ZJP could certainly boost the green uorescence and reduce the red uorescence of GES-1 cells (Fig. 3B-D).These results indicated that ZJP could ameliorate nuclear morphology and cell proliferation in H. pylori-induced injury.

JMJD2B/COX-2/VEGF axis mRNA and protein expression in Vivo
Previous gene ontology analysis revealed that JMJD2B regulates VEGF signaling pathways and angiogenesis which play important roles in in ammation or cancer progression [14].Blood circulation disorders signi cantly in uence pathological process of CAG.VEGF is the target gene to closely regulate angiogenesis, which can stimulate the proliferation of epithelial cells, the formation of blood capillaries, and then participating in the defense and repair of gastric mucosa.Widely accepted, COX-2 is a prostaglandin-endoperoxide synthase, which is responsible for the formation of thromboxanes as a key rate-limiting enzyme.In H. pylori-infected gastric mucosal cells, COX-2 is involved in the regulation of VEGF expression [20].Nevertheless, whether ZJP could interfere with CAG through JMJD2B/COX-2/VEGF axis has not been studied.IHC revealed that Control group samples expressed low levels of JMJD2B as evidenced by barely positive staining of JMJD2B in gastric mucosal cells.Correspondingly, model group samples hold higher level of COX-2 compared with non-infected tissues.Interfered by ZJP, the expression of JMJD2B and COX-2 decreased (Fig. 4).
Compared with control group, the serum TNF-a level was signi cantly increased in model group.After administration of ZJP group (0.63, 1.26 and 2.52 g/kg), TNF-α signi cantly reduced, and the high dose group (2.52 g/kg g) and Omeprazole group had lower level of TNF-α compared with group with low dose group and medium dose group (Fig. 5F).Moreover, RT-qPCR and Western Blot were used to explore intervention effect of ZJP on JMJD2B/COX-2/VEGF axis (Fig. 5B-K).Compared with control group, JMJD2B, COX-2, VEGF, VEGFR1 and VEGFR2 expressed at a relatively high level in model group.However, the mRNA and protein expression levels of these genes in the ZJP groups were decreased.High dose group of ZJP could signi cantly reduce the mRNA and protein expression levels, while, the medium and low dose group of ZJP exhibited weaker reduction.

JMJD2B/COX-2/VEGF axis mRNA and protein expression in vitro
We previously demonstrated that the mRNA and protein levels of JMJD2B, COX-2, VEGF, VEGFR1 and VEGFR2 were signi cantly decreased following intervention of ZJP.In order to further explore the effect of ZJP on JMJD2B/COX-2/VEGF axis, the model of H. pylori induced (MOI=50:1, 12h) GES-1 cells was established (Fig. 6).The IL-8 mRNA level was signi cantly increased in H. pylori-infected cells.ZJP at 30 μg/mL and 60 μg/mL could all decrease the IL-8 mRNA level compared to control group (Fig. 6G).
Evidently, the expression of JMJD2B, COX-2, VEGF, VEGFR1 and VEGFR2 were increased in H. pyloriinfected cells compared with the control group.
Administration of ZJP at high dose (60 μg/mL) expressed lower level of JMJD2B and its downstream genes noticeably compared with the H. pylori-infected group and low dose (30 μg/mL) group.The results further emphasize that ZJP may relieve H. pylori-induced in ammation and gastric mucosa injury via the downregulation of JMJD2B/COX-2/VEGF axis.

Discussion
In this study, results revealed that ZJP could suppress the JMJD2B/COX-2/VEGF axis to exhibit antiin ammatory properties in H. pylori-induced CAG.It could attenuate the gastric mucosal injury by suppressing the in ammatory factor via inhibiting COX-2 and VEGF expression, which is involved in the anti-in ammatory effect of ZJP in CAG with H. pylori induction.Our study set the foundation to explore the mechanism and pharmacological effects of ZJP in the treatment of H. pylori-induced CAG.
H. pylori is a Gram-negative bacterium and colonizes approximately over 50% of the world's population, with a variable prevalence in different regions.H. pylori was classi ed as a class I carcinogenic factor by the International Agency for Research on Cancer in 1994.H. pylori is a major human pathogen causing progressive, chronic gastric mucosal injury and linked to CAG and gastric cancer.Epigenetics plays the vital role in the development and progression of gastric cancer [21].H. pylori infection induces epigenetic changes, like DNA methylation and histone modi cation, which play important roles in oncogenic transformation [22].Histone modi cation, an epigenetic mechanism, which plays a crucial role in GC carcinogenesis [23].The family of JMJD2 consists of JMJD2A, JMJD2B, and JMJD2C.JMJD2B speci cally catalyzes the removal of di-and trimethylated H3K9 (H3K9me2/me3), converting both histone marks to the monomethylated state [24].Thus, JMJD2B plays a role as a transcriptional activator [24].Previous studies have discovered that H. pylori infection can affect JMJD2B expression in gastric epithelial cells, which is involved in the pathological development of gastric cancer [25].As we all know, COX, as a key rate-limiting enzyme, promotes arachidonic acid convert to prostanoids and thromboxanes, which has two forms, COX-1 and COX-2.COX-1 maintains normal function in most tissues.In contrast, COX-2 associated with pain, in ammatory reaction, tumorigenesis and so on.Besides, the expression of COX-2 is known to be increased in the gastric mucosa of H. pylori-infected gastritis patients [26].In H. pylori-infected gastritis, there is an increase in angiogenic factors, and subsequently a formation of new blood vessels.New angiogenesis will enhance supply of nutrient and oxygen, and promote the development of gastritis [27].Gastritis induced by H. pylori is related to VEGF, and the overexpression of VEGF is parallel to the increase in gastric mucosal vascularization [28].VEGF is the key regulator of in ammation-related angiogenesis and overexpression in gastric diseases induced by H. pylori [29].On the one hand, VEGF involved in the construction of normal mucosal.On the other hand, it also accelerates gastric carcinomas by supporting tumor-associated angiogenesis [30].COX-2 induced the overexpression of VEGF in gastric tissues colonized by H. pylori [33].H. pylori infection might be able to induce the expression of COX-2 in gastric tissue, which in turn upregulates the expression of VEGF [31].In this present study, we veri ed the role of the JMJD2B/COX-2/VEGF axis in H. pylori induced CAG.
In this current study, the effect of JMJD2B/COX-2/VEGF axis is veri ed one more time in vitro and in vivo.
The role of the axis in the promotion of gastric mucosa damage and in ammatory responses has been described.Consequently, it is potential that activation of JMJD2B/COX-2/VEGF axis may accelerate the process of chronic infection or even has high tendency of canceration.In this study, the expression of proin ammatory genes, including JMJD2B, COX-2, VEGF, VEGFR1, VEGFR2, and TNF-a were probed in SD rats when H. pylori infection.We found that the expression of TNF-a was activated in H. pylori induced rats.In addition, genes in JMJD2B/COX-2/VEGF axis also increased after H. pylori infection.Next, the genes related to JMJD2B/COX-2/VEGF axis were quested in the H. pylori-infected cells.Accordingly, the expression of JMJD2B, COX-2 and downstream genes including VEGF, VEGFR1 and VEGFR2 were increased signi cantly in H. pylori-infected cells.We also revealed the protein expression in JMJD2B/COX-2/VEGF axis got escalated in H. pylori-infected GES-1 cells and gastric mucosa.There is no denying that JMJD2B/COX-2/VEGF axis exerts pivotal effects in chronic in ammation process induced by H. pylori.
In China, Chinese medicine and prescriptions based on the theories of TCM have been considered.TCM, as a complementary therapy, has achieved vital effects in treating CAG and broadened the ideas of therapeutic approaches for CAG [32].ZJP has been used for the treatment of gastrointestinal disorders in the clinical practice of TCM more than 600 years.According to the current o cial Chinese Pharmacopoeia, ZJP has been prescribed as the effective prescription of gastritis, pyloric obstruction, gastric ulcer, gastroesophageal re ux disease, etc.In this study, we explored the pharmacological effects of ZJP in the treatment of H. pylori-induced CAG.The mechanism of ZJP in treating H. pylori-infected CAG were also explored by analyzing the JMJD2B/COX-2/VEGF axis.In our examination, we found that ZJP administration signi cantly improved the cell protection from H. pylori infection.Cell viability, morphological identi cation of GES-1 cells was directly ameliorated following ZJP administration.The pathological changes of gastric mucosa, including arrangement of gastric glands, in ltration of lymphocytes and plasma cells, were also better than model group.The speci c marker of serum TNF-a signi cantly decreased after intervened by ZJP.We speculated that with H. pylori activity intervened by ZJP, the in ammation level and damage of gastric mucosa returned to normal levels.In terms of mechanism of ZJP in treating H. pylori-infected CAG, the proin ammatory genes, encoding JMJD2B, IL-8, COX-2, VEGF and its downstream genes were either signi cantly downregulated by ZJP intervention in GES-1 cells.These results support the role of ZJP as an anti-in ammatory agent in GES-1 cells damage induced by H. pylori.We also demonstrated the expression of JMJD2B/COX-2/VEGF axis including JMJD2B, COX-2, VEGF, VEGFR1 and VEGFR2 were signi cantly suppressed in H. pylori-infected gastric mucosa.Meanwhile, we revealed the protein expression in JMJD2B/COX-2/VEGF axis was downregulated after exposure to ZJP.Taken together, these results suggested that ZJP which could block the course of JMJD2B/COX-2/VEGF axis to H. pylori-infected procession in vivo and in vitro.

Conclusions
Taken together, our study con rmed the therapeutic effect of ZJP in H. pylori-induced CAG model.We also found that Histone demethylase played a vital role in CAG model.Importantly, ZJP prevented gastric mucosal injury by inhibiting the H. pylori-mediated in ammation via JMJD2B/COX-2/VEGF axis.The results from this study suggest a potential role of ZJP in treatment of CAG, which need to be further investigated.

Declarations
Table

Figure 2 Cell
Figure 2

Figure 3 High
Figure 3

Figure 6 Effects
Figure 6

Figure 7 Effect
Figure 7

Figure 8 Effects
Figure 8

Figure 9 Effects
Figure 9 control group were induced with equal volume saline by oral gavage.All rats were fasted about 12 h before intragastric administration.After 8 weeks, gastric tissues were obtained for rapid urease test to detect the model.Finally, the CAG rats were randomly divided into ve different groups with six rats in each, including the model group, ZJP low-dose (0.63 g/kg), medium-dose (1.26 g/kg) and high-dose (2.52 g/kg) groups, and Omeprazole group (1.8 mg/kg).All rats were administered once a day for 4 weeks.After 4 weeks, all rats were executed and gastric mucosa samples were isolated and cut in half along the greater curvature.The serum and half of the gastric tissue samples were collected and stored at -80°C to detect expression of gene and protein.The other of the gastric tissue samples were excised and xed in 4% paraformaldehyde general tissue xative, and then stained with H&E.