Investigation Of Heat Shock Protein 70 In The Pathogenesis Of Chronic Atrophic Gastritis

Gastric carcinoma (GC) remains the second most common cause of cancer death worldwide with approximately 870,000 new cases and 650,000 deaths per year. There is no efficient method for early diagnosis or therapy, and the five-year survival rate for most GC patients is less than 20%. So it has been suggested to prevent gastric carcinoma by intervention or reversion of the premanlignant diseases.According to the Correa's cascade, gastric cancer was supposed to develop in a multistep progression from chronic atrophic gastritis (CAG), intestinal metaplasia (IM), to dysplasia and finally intestinal-type adenocarcinoma. In 1998, gastrointestinal pathologists recognized the close correlation between CAG and gastric cancer and indicated CAG as a pre-malignant lesion. CAG is characterized with chronic inflammation of the gastric mucosa and loss of gastric glandular cells with replacement by intestinal-type epithelium, pyloric-type glands, and fibrous tissue. It has been reported that about 1% to 7.46% of CAG developed into GC. So it is necessary to investigate the underlying mechanism of CAG for the further prevention of gastric carcinogenesis. Till to now, little is known about the molecular mechanisms of CAGHeat shock protein70 (HSP70), as intracellular molecular chaperon, could accelerate the cellular recovery from different stimuli by cope with unfolded or denatured proteins, interacts with immune system of cells and exerts immunoregulatory effects. HSP70 also plays an important role on regulating apoptosis and proliferation. Recent studies have reported that HSP70 exerts cytoprotective role in some gastropathy, such as gastric ulcer and portal hypertensive gastropathy, and it has been proposed that inducing HSP70 protein might provide a therapeutic strategy for protection against mucosal injury. However, little literature has shown the correlation between HSP70 and CAGWhy we proposed that HSP70 is possible to involve in the pathogenesis of CAG originated from the following reasons: 1) CAG is the results of chronic stresses, and aberrant expression of HSP70 could impaired cellular stability and enhanced susceptivity of cells to injuries; 2) Some studies indicated that the deranged apoptotic and proliferative signaling, as well as the following acquisition of genetic mutations could be associated with the progression of CAG and carcinogenesis. HSP70 is indicated to posses the ability of regulating the balance between apoptosis and proliferation, as well as promoting degradation of mutated proteins; 3) H.pylori is defined as I type risk factor for CAG and gastric carcinoma. A latest study reported that H.pylori infection decreased the levels of HSP70, which might limit the host inflammatory response and facilitate chronic infection by avoiding host immunity. So to investigate the correlation between HSP70 and CAG would possibly provide a new insight into the formation of CAG and possibly explore a targeted factor for CAG treatment. Therefore, Our study was designed to investigate the expression of HSP70 in CAG and to evaluate the possible role of HSP70 in the pathogenesis of CAG, which was consist of following parts: 1) to analyze the expression of HSP70 in patients with chronic atrophic gastritis; 2) to evaluate the role of HSP70 in the progression of CAG in rats after treatment with HSP70 inducer-GGA or HSP70 inhibitor-quercetin; 3) to investigate the effects of HSP70 on gastric epithelial cells line AGS by HSP70 inhibitor-quercetin and siRNA technology; 4) to explore the involvement of HSP70 in the pathogenesis of H.pylori in gastric epithelial cells..Material and MethodsSection I: To investigate the association of HSP70 and chronic atrophic gastritis, we assessed the expression of HSP70 in gastric samples from patients with chronic atrophic gastritis. Gastric biopsy specimens were obtained from 59 patients who underwent gastroendoscopy and have given consent for additional biopsy, including 43 with chronic atrophic gastritis and 16 normal controls. The distribution and the level of HSP70 expression were measured with immunohistochemistry and western bolt. The correlation of HSP70 and chronic atrophic gastritis was further analyzedwith mono-variance analysis and multiple regression analysis.Section II: To investigate whether HSP70 would involve in the progress of CAG, we evaluated the pathological alterations with the treatment of HSP70 inducer (GGA) or inhibitor (quercetin) in rats with CAG In brief, male mature Sprague-Dawley rats were induced CAG by the "S"-method. After 24 w of the integrative induction, rats were administered with GGA, quercetin or solvent control (DMSO) for another 4 w respectively. HSP70 expression was evaluated by Western blot and immunohistochemistry. Histological assessment of gastric antrum was in accordance with the diagnostic criteria of gastritis in Houston in 1994. The count of infiltrated inflammatory cells, the thickness of the mucosa glandular layer (μm) and the quantity of gastric glands in 1mm horizontal length were calculated.Section III: To elucidate the effect of HSP70 on gastric epithelial cells line AGS, we detected the alterations of apoptosis and proliferation by HSP70 inhibitor, quercetin and HSP70siRNA interference respectively. After incubation AGS with different concentrations of quercetin, we assessed the proliferation and apoptosis by means of MTT, flow cytometer and TUNEL assay. The expressions of apoptosis-associated proteins including Bax,Bcl-2,AIF and cleaved Caspase-3 were evaluated by western blot. Further investigation was done with the small RNA interference technology. AGS cells were transfected with the vector of siRNA-HSP70 and stable HSP70siRNA-transfected AGS was established by the selection of G418. The growth curve and cell cycle of HSP70siRNA-transfected AGS cells, as well as the expression of apoptotic proteins were assessed.Section IV: To evaluate the involvement of HSP70 in the pathogenic mechanism of H. pylori infection, we incubated HSP70siRNA/AGS with H. pylori and observed the variance of cells viability and cells cycle in contrast with the empty vector control. Furthermore, exogenous HSP70 (50ng/ml) was added into the incubation. Apoptotic proteins including Bax, cytochrome c, caspase-3, caspase-6, caspase-7 and AIF were assessed by western blot.Results1. HSP70 expression was found to fairly decrease in patients with atrophy and intestinal metaplasia in contrast with normal controls by both mono-variance and multiple regression analysis.2. CAG was successfully induced in rats by "S"-methods, which showed that moderate neutrophil cells and lymphocytes infiltrated in gastric mucosa (inflammation index: 1.51±0.17 in CAG vs. 0.88±0.24 in normal control, P<0.001). The glandular layer was attenuated (135.85±46.50 μm in CAG vs. 234.00±42.33 μm in normal control, P<0.001) and the glands in gastric antrum arranged mussily and sparsely (quantity of glands:37.41 ±4.93/mm in CAG vs. 46.80 ±2.85/mm, P<0.05). Western blot analysis revealed a decreased HSP70 expression in CAG rats. Comparing with control, treatment with GGA resulted in a rapid relief of inflammation (inflammation index: 1.1+0.43 vs. CAG, P<0.05) and the restoration of glandular cells (the thickness of glandular layer: 187.22+27.50μm, vs. CAG P<0.05 and glands count: 41.44 + 3.90/mm, vs. CAG P>0.05), as well as a considerable induction of HSP70. In contrast, quercetin induced a notable mucosal congestion and an appreciably increase in the infiltration of inflammatory cells (inflammation index: 1.80+0.33 vs. CAG, P<0.05). The synthesis of HSP70 protein was inhibited in gastric mucosa by treatment with quercetin.3. Quercetin with the concentrations between 50-300μM inhibited the HSP70 expression in a dose-dependent manner in AGS cells. Furthermore, it suppressed proliferation with the depressed viability of cells and S-phase blocking. Quercetin induced apoptosis in the dose- and time-dependent manners, which was concomitant with the increase of proapoptotic Bax, AIF and cleaved caspase-3, but the decrease of antiapoptotic Bcl-2.4. HSP70siRNA/AGS cell was successfully established, whose HSP70 expression was depressed by 45%+15%. Comparing with the empty vector control, HSP70siRNA/AGS cell was observed a retardance of growth, with increasing vacuolar cells and S-phase blocking. The elevated expression of Bax, AIF and cytochrome c in cytosol was observed.5. H. pylori (ATCC700392) facilitated the proliferation of gastric epithelial cells in the early phase of incubation and inhibited the viability of cells in the later. Furthermore, the inhibitive effect of H.pylori on proliferation was enhanced by HSP70 downregulation in contrast with the control cells. Inhibition of HSP70 in AGS cells further induced the release of Cyto c from mitochondria to cytosol, up-regulated AIF expression and decreased the level of pro-caspase-3. Exogenous HSP70 reduced the release of cytochrome c from mitochondria, but increased the expression of AIF.Conclusions:1. HSP70 expression was reduced in CAG that was testified in both clinical and animal investigations. HSP70 inducer-GGA facilitated the relief of inflammation and glandular restoration in gastric mucosa of rats with CAG; However, HSP70 inhibitor-quercetin aggravated the infiltration of inflammatory cells in CAG rats, which urgently suggested the possible involvement of HSP70 in the pathogenesis of CAG.2. Quercetin inhibited HSP70 expression and depressed the viability of AGS cells. Moreover, S-phase blocking and apoptosis induced by quercetin further suggested the regulatory effect of HSP70 on proliferation and apoptosis in gastric epithelial cells, which could be associated with the mechanism of HSP70 in CAG3. Stable cells transfected with siRNA-HSP70 was established successfully. HSP70 downregulation retarded the growth of gastric epithelial cells and blocked cell cycle at S-phase, suggesting HSP70 inhibition attenuated the self-stability of gastric epithelial cells. The aberrant expression of apoptotic proteins including Bax, AIF and Cyto c further confirmed the important effect of HSP70 on regulating growth and apoptosis in gastric epithelial cells.4. H.pylori facilitated the proliferation of AGS cells in the early phase of incubation and inhibited the viability of cells in the later, which was independent of HSP70 expression. However, suppression of HSP70 by siRNA aggravated the inhibitive effect of H. pylori on cells viability. The results suggested HSP70 was involved in the pathogenesis of H. pylori infection.