Protective Effect of Zuojin Pill on Helicobacter Pylori-Induced chronic atrophic gastritis in Rats and GES-1 Cells and Mechanisms of Action Exploration

Objective Zuojin Pill (ZJP) containing two Chinese is a classical formula and is widely accepted as a treatment of chronic atrophic gastritis (CAG) in China. This study aimed to explore the therapeutic effect and mechanism of ZJP which attenuated H. pylori -induced CAG in vivo and in vitro. Methods: H. pylori (Helicobacter pylori) was used to induce CAG rat model. 0.63, 1.26, and 2.52 g/kg of ZJP (was administered orally for four weeks. Therapeutic effect of ZJP was identified by H & E staining and serum indices. In addition, cell viability, morphology and proliferation were detected by cell counting kit-8 and high-content screening assay. Gene and protein expression related to JMJD2B/COX-2/VEGF axis were detected to further investigate the potential mechanism. Results Compared with the control group, the ZJP groups showed a significant protection effects on Gastric mucosa, as indicated by the reduced loss of glands and inflammatory cell infiltration. Meanwhile, ZJP could ameliorate cell viability, morphology changes, and proliferation in GES-1 cells. Moreover, the ZJP treatment decreased the amount of IL-8, and TNF-α, indicating that it could reduce the level of inflammation, and decrease stomach damage. The expression of JMJD2B/COX-2/VEGF axis related genes and proteins were measured by real-time quantitative PCR, western blot and immunohistochemistry methods. The ZJP groups were found to decrease relative genes and protein expression level compared with the model group. ZJP could improve gastric mucosa protection and reduce inflammation level by inhibiting the expression level of JMJD2B/COX-2/VEGF axis. Conclusion Our data confirmed the effective therapy of ZJP in H. pylori -induced CAG, which supports the role of ZJP as an anti-inflammatory and protection of gastric mucosa agent in CAG induced by H. pylori . These results may provide helpful tools for the treatment of CAG.

enough food and water. All specific pathogen free (SPF) male SD rats (170-190g) were purchased from Beijing Sibeifu Animal Breeding Center [Permission No. SCXK-(Jing) 2016-0002]. Firstly, the rats were randomly divided into the control group and model group. The rats in the model group were induced with H. pylori (1.5×10 8 CFU/ml, 1.5 ml each rat) suspension to establish CAG model (4 times a week, at day 1, 3, 5 and 7) and rats in the control group were induced with equal volume saline by oral gavage. All rats were fasted about 12 h before intragastric administration. After the infection for 8 weeks, gastric tissue was obtained for rapid urease test to detect confirm the model. Finally, the animals with successfully prepared CAG model were randomly divided into five 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 (1.8mg/kg). Rats in all groups were administered with corresponding drugs once a day as long as 4 weeks including the control group. After 4 weeks, all rats were executed and gastric mucosa samples were isolated and cut in half along the greater curvature, and then were rinsed with Saline. The serum and half of the gastric tissue samples of each rat were collected and stored at -80°C for the detection of mRNA and protein expression. The other of the gastric tissue samples was excised and fixed in 4% paraformaldehyde general tissue fixative, and then stained with hematoxylin and eosin (HE).

Serum Tumor Necrosis Factor -α (TNF-α) and VEGF measurements
The serum TNF-α and VEGF levels 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 cells were cultured overnight to reach at least 80% confluency. 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). The number of GES-1 cells was obtained through counting slides (Bio-Rad, USA). Cocultivation was maintained at 37°C in a 5% CO 2 atmosphere.

High-Content Analysis Experiments (HCS)
Nuclear, cell morphology and number of dead cells and living cells were detected by using the Array Scan High-Content System (Thermo Scientific, Massachusetts, USA) [10]. 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 profiling assay module was selected in the HCS system, and several different wavelength channels were set to collect fluorescence images. The measured parameters and format were similar to those used previously [11]. Array Scan XTI (The Array Scan software algorithm was used to perform analysis) was used to quantify the mean fluorescence 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 using TRIzol reagent (Nordic Bioscience, Beijing, China) and transformed into cDNA by using 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 βactin 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 significant when P < 0.05 and highly significant when P < 0.01.

Results
ZJP ameliorates macro performance of H. pylori-induced chronic atrophic gastritis in SD rats Firstly, through 8 weeks of H. pylori induction, the rapid urease test reaction was used to confirm the CAG rat model. The rapid urease reaction in the gastric antrum of rats in the model group was positive with red light, while the control group was negative with yellow color (Fig. 1A). After a 4-week administration, the reaction in the ZJP low-dose group was negative compared with the model group ( Fig. 1B). During the 8 = week H. pylori induction, the weight of rats in the model group showed a downward trend. After a 4-week administration, the weight of the rats gradually increased (Fig. 1C).
The gastric mucosa of rats in model group showed paleness and thinning of gastric mucosa, with disarrayed plicae and small white nodules, while was pink, moistened and smooth in the control group. The rats in the ZJP low-dose group exhibited good elasticity, mild mucus conjugation and edema on the mucosal surface, dark color and regular folds (Fig. 1D).

ZJP ameliorates H. pylori-induced Histological examination of gastric damage in SD rats
Histological features of gastric tissue were the critical evidence for the therapeutic effect of ZJP against H. pylori-induced CAG. In this study, H & E staining was used to evaluate the loss of glands and inflammatory cell infiltration in gastric tissue of rats (Fig. 2). Rats in the control group showed Mucosal intact with tightly, abundant and orderly gastric glands. In the model group, rats showed inherent glands and that in the gastric tissue was missing, part of the mucosa was stripped, and lymphocytes and neutrophils were infiltrated in the mucosa. Conversely, gastric tissue in the omeprazole and the ZJP high-dose group, pathological changes were significantly lower than in the model group in terms of the degree of edema, hyperemia, erosion and atrophy of gastric mucosa was significantly alleviated Administration of ZJP medium-does group and low-does group exhibited a moderately reduced severity of inflammatory cell infiltration and other histological injuries.
Effects of ZJP on the proliferation of GES-1 cells Cell viability was detected by using CCK-8 kit to determine the appropriate concentration of ZJP to GES-1 cells (Fig. 3). The results showed that ZJP treatment for 24 h potently suppressed cell viability in a concentration-dependent manner with increasing the concentration (0, 10, 20, 30, 40, 60 and 120 µg/ml), among which, 120 µg/ml of ZJP could significantly inhibit the cell viability compared with the control group (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 on GES-1 cells. 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.  (Fig. 5B-C). These results indicated that ZJP could ameliorate nuclear morphology and cell proliferation in H. pylori-induced injury and cytotoxicity in GES-1 cells.
ZJP reduced the Serum TNF-a Level in CAG rats and IL-8 mRNA expression in H. pylori infected cells The serum supernatant of TNF-α was measured to elucidate the expression level of TNF-α in H. pyloriinduced CAG rats. Compared with control group, the serum TNF-a level was significantly increased in H. pylori-infected rats. ZJP at 0.63, 1.26 and 2.52 g/kg could all decrease the serum TNF-a level in a dose-dependent manner (Fig. 6A). Omeprazole could also obviously decrease the TNF-a level. The IL-8 mRNA level was significantly increased in H. pylori-infected GES-1cells. ZJP at 30 µg/mL and 60 µg/mL could all decrease the IL-8 mRNA level compared to control group (Fig. 6B).

ZJP induce JMJD2B and COX-2 expression in H. pylori-infected animal models
Recently, emerging evidence has shown that H. pylori could promote the integration of JMJD2B with COX-2 promoter and then recruit NF-κB to bind on COX-2 promoter, and further to improve COX-2 induction [8]. Here, we explored whether ZJP could reduce gastric mucosa injury via regulating protein expression of JMJD2B and COX-2. IHC was used to determine those expression levels ( Fig. 7A-B). The model group showed boosted levels of JMJD2B and COX-2, compared with expression levels in the control group. However, ZJP treatment can obviously decrease those protein expressions. This phenomenon provided the first evidence that ZJP may relieve gastric mucosa injury via the downregulation of JMJD2B and COX-2 activity.

ZJP induced JMJD2B/COX-2/VEGF axis mRNA and protein expression in Vivo
Blood circulation disorders significantly influence 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 [12]. Nevertheless, whether ZJP could interfere with CAG through JMJD2B/COX-2/VEGF axis has not been studied. In the present study, the mRNA and protein expression levels of JMJD2B, COX-2, VEGF, VEGFR1, and VEGFR2 in the model group were significantly elevated compared with the control group (Fig. 8). Compared with the model group, the mRNA and protein expression levels of these genes in the ZJP groups were decreased. High-dose group of ZJP could reduce the mRNA and protein expression levels, while, the medium-and low-dose group of ZJP exhibited weaker reduction.

ZJP inhibited JMJD2B/COX-2/VEGF axis mRNA and protein expression in vitro
The model of H. pylori induced (MOI = 50:1, 12 h) GES-1 cells was established to further confirm the role of ZJP for relieving H. pylori infected gastric epithelial cell damage through JMJD2B/COX-2/VEGF axis in vitro (Fig. 9). Firstly, the mRNA expressions of JMJD2B, COX-2, VEGF, VEGFR1 and VEGFR2 in GES-1 cells were measured. Consistent with the expression of mRNA and protein in rats, the mRNA and protein levels of JMJD2B, COX-2, VEGF, VEGFR1, and VEGFR2 were significantly increased after the infection of H. pylori and could decrease after treating with ZJP to some degree. This phenomenon further strengthened that ZJP may relieve H. H. pylori-induced inflammation and gastric mucosa injury via the downregulation of JMJD2B/COX-2/VEGF axis activity.

Discussion
The clinical features of CAG are focused on satiety, belching, abdominal pain and nausea, and weight loss. CAG is a proverbial signal of precancerous lesions of gastric cancer, which has currently been the second most common cause of cancer-related deaths worldwide [13]. In rodent models, H. pylori gavage causes a series of inflammatory reactions in the gastric mucosa, such as inflammatory cell infiltration [14]. Thus, in this study, H. pylori was used for preparing the CAG model in rats in this study to investigate the intervention effect and mechanism of ZJP in vivo and in Vitro.
In the CAG rat model group, the weight of rats, H. pylori colonization and inflammatory factor, as well as gastric pathological features were ameliorated after treatment with ZJP. Histological analysis showed that loss of glands and inflammatory cell infiltration were reduced to a certain extent after ZJP treatment. In addition, ZJP ameliorated cell viability, morphology changes and proliferation in GES-1 cells. On the basis of the above results, ZJP had the potent possibility to prevent the development of CAG.
Epigenetics plays the vital role in the development and progression of gastric cancer [15]. H. pylori infection induces epigenetic changes, like DNA methylation and histone modification, which plays important roles in oncogenic transformation [16]. JMJD2B can promote the occurrence and development of gastric cancer and serves as a potential biomarker in gastric cancer [17]. Recently, emerging evidence has shown that H. pylori could promote the integration of JMJD2B with COX-2 promoter and then recruit NF-κB to bind on COX-2 promoter, and further to improve COX-2 induction [8]. Here, we next explored whether ZJP could reduce gastric mucosa injury via regulating protein expression of JMJD2B and COX-2. 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 prostaglandin-endoperoxide synthase, which 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 [12].
Therefore, controlling JMJD2B/COX-2/VEGF axis might be effective in the treatment of CAG.
COX, as is a key rate-limiting enzyme, can promote arachidonic acid convert to prostanoids and thromboxanes in two forms, COX-1 and COX-2 [18]. COX-1 maintains normal function in most tissues.
In contrast, COX-2 associated with pain, inflammatory 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 [19]. In H. pylori-infected gastritis, there is an increased 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 [20]. H. pylori-induced gastritis is associated with VEGF, whose overexpression parallels the increased formation of blood vessels in the gastric mucosa [21]. COX-2 could induce overexpression of VEGF in the gastric tissue colonized by H. pylori. H. pylori infection might be able to induce the expression of COX-2 in gastric tissue, which in turn upregulates the expression of VEGF [22]. In this study, it is found that the expressions of VEGF and its receptor VEGFR1 and VEGFR2 were decreased remarkably after treatment with ZJP both at mRNA and protein level, suggesting that ZJP could decrease the expression of VEGF/VEGFR1/VEGFR2. Furthermore, the level of JMJD2B was higher in H. pylori group than control group, while was reduced notably after treatment with ZJP. In H. pylori-infected cells, the expression of JMJD2B gene was increased with increasing the infection time and plural number in a certain range. In this examination, Proinflammatory genes, IL-8 mRNA expression and TNF-a protein levels were elevated significantly, showed that, compared with the H. pylori group, ZJP could reduce the expression of IL-8 and TNF-α. In conclusion, ZJP might improve inflammation in CAG rats by inhibiting JMJD2B/COX-2/VEGF axis.
Based on the above data, ZJP was confirmed to improve CAG and gastric mucosal injury induced by H.
pylori. In addition, the results of the present study suggested that JMJD2B/COX-2/VEGF axis is closely related to the therapeutic and anti-inflammatory effects of ZJP on CAG. JMJD2B/COX-2/VEGF axis plays an important role in H. pylori induced inflammation. These results of this study laid a theoretical foundation for the further study of ZJP in the treatment of chronic atrophic gastritis.

Conclusions
Taken together, our study confirmed 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 inflammation 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.