Mitochondrial Damage Induced By Helicobacter Pylori And Its Drug Intervention

BACKGROUND AND AIMS: Gastric carcinogenesis is a multistep process and a multifactorial related event, in which the role of Helicobacter pylori (H.pylori) infection involved has been established in recent years. It was found that apoptosis induced by H. pylori plays an important role in the gastric carcinogenesis. Mitochondria is thought to be the apoptosis-inducting center in mammalian cell. The apoptosis process can be divided into three stages: pro-mitochondrial stage, mitochondrial stage and post-mitochondrial stage. The pro-mitochondrial stage is associated with various kinds of stimulus for apoptotic signal transduction. The mitochondrial stage is the central modulation step of initiation apoptotic pathway. The post-mitochondrial stage includes a series of hydrolytic enzymes release. Mitochondrial permeability transition (MPT) is the crucial process in the mitochondrial stage, which is served as the switch to cell surviving or death. The impairment or loss of mitochondrial membrane potential (△Ψm) in MPT may result in that the cell goes into the apoptotic pathway. The Lengthwise study of the changes of mitochondrial morphologies,structures and functions in the gastric carcinogenesis induced by H. pylori along with the time course of apoptotic-genesis has not been reported so far. On the other hand, it is not clear if the simple eradication of H. pylori could reverse gastric precancerous lesions induced by this bacteria, and that need to be further studied. Even though it is not widely applied in the clinic, the vaccination of H. pylori might be an ideal strategy in the prevention of its related gastric carcinoma.Epidemiological and prospective study have shown that specific inhibitors of cyclooxygenase-2 may inhibit the growth of some cancer cells (e.g. colorectal , esophageal and hepatic carcinoma), however, its effects on the H. pylori correlated gastric carcinoma has not been investigated. In this study, we carried out the following four parts of experiment: 1. To establish the animal model of H.pylori infected Monglial gerbils (MGS), and further to observe the pathological changes in the gastric mucosa; 2. To observe the changes in mitochondrial morphology,function and the expression of its mRNA and encoding proteins induced by H. pylori in gastric epithelia cell in vitro; and 3. To evaluate the effects of H. pylori infection on mitochondrial morphology and function, and expression of apoptosis-related protein in vivo; 4. To investigate the effects of celecoxib on gastric carcinoma and its mechanisms in vitro and in vivo. This study consists of four parts: 1. To establish the animal model of H. pylori infected Monglial gerbils(MGS) and observe the pathological changes ; 2. To observe the changes of mitochondrial morphology,function and its encoding protein and mRNA induced by H. pylori in vitro; 3. To investigate the changes of mitochondrial morphology,function and apoptotic protein by H. pylori in vivo; 4. To evaluate the effects of celecoxib on gastric carcinoma and mechanisms in vitro and vivo. METHODS: 1. 120 MGS were randomized into five groups according to the H. pylori infection and/or compound treatment (24 for each group). Group A, H. pylori; Group B, H. pylori +MMNG; Group C, MMNG; Group D, H. pylori +MMNG +celecoxib; Group E, which was negative control. Eight animals were sacrificed in each group after 12, 24, 48 experimental week. The following observations were performed: (1)The changes of animal body weights; (2) The H. pylori infection was detected by Urease enzyme test, Warthin-Starry silver staining and polymerase chain reaction (PCR) of H. pylori urease gene A (ureA); (3) The pathology changes of gastric mucosa by microscopy. 2. H. pylori strains NCTC 11637 were co-cultured with human gastric adenocarcinoma epithelial cell lines SGC-7901 cells. We further performed these experiments: (1) To observe gastric mucosa microstructure changes by electron microscopy; (2) Expressions of Cyt-c, Bcl-2 and Caspase-9 were determined by immunohistochemistry staining; (3) Apoptotic rates, the concentrations of Ca2 + and the levels of △Ψm were assessed by Flow cytometry; (4) Semi-quantitative RT-PCR was used to measure the mRNA expression of mtDNA COXI,COXII and COXIII; (5) The mtDNA COXI protein expression was determined by Western analysis. 3. 48 MGS were randomized into two groups (24 for each group). Group A, H. pyloriinfection; Group B, which was negative control. Eight animals were sacrificed in each group after 4,12,24 experimental week. We performed another observations as mentioned above: (1) The changes of animal body weights; (2) The detection of H.pylori infection; (3) The gastric mucosa pathology and microstructure changes; (4) The immunochemistry staining of Cyt-c, Bcl-2 and Bax; (5) Apoptotic rates, Ca2 + concentrations and △Ψm. 4. 10 SPF BALB/C-nu/nu nude mice underwent the tumor implantation study. After celecoxib treatment in SGC-7901 cells, the cell proliferation was detected by MTT method. Cell cycle,apoptotic rates , Ca2 + concentration and △Ψm were assessed by Flow cytometry. SGC-7901 cells divided into four groups. Group A was control; Group B, H. pylori infection; Group C, celecoxib treatment, Group D, H. pylori + celecoxib. (1) The cyclooxygenase-2 mRNA expression was measured by semi-quantitative RT-PCR; (2) The proteins expression of cyclooxygenase-2 and Caspase-9 was determined by Western analysis. RESULTS: 1.Pathological changes: In group A, Chronic atrophy gastritis were observed in 15 MGs, intestinal metaplasia were found in eight animals, displasia in nine, and in situ carcinoma in one. Histopathological changes showed grandular structures and cells displasia in the whole mucosa layers, nuclear enlargement and prominent nucleoli, high nuclear/cytoplasmic ratio, the irregular nuclear outlines ,and the uneven distribution of chromatin, with grandular strctures irregular. In group B, fifteen animals showed chronic atrophy gastritis, five showed intestinal metaplasia, thirteen showed displasia. In group C, nine animals showed chronic atrophy gastritis, two showed slight displasia. In group D, four animals showed chronic atrophy gastritis, one showed intestinal metaplasia. In group E, there were no pathological changes noted. In groups A and B, the incidence rates of intestinal metaplasia and dysplasia were higher than groups C , D and E ( P<0.05). 2. Experiment in vitro: (1).Swelling intramitochondrial compartments, or reduced cristae or disappearing outmembrane were observed in cells after exposure to CHCS for 12 h, when treated with CHCS for 24 h , and the apoptotic body released . (2).Immunohistochemistry showed weak positive staining of Cyt-c and Caspase-9 proteins in the SGC-7901 cells. Expression was increased in the group after exposure toCHCS for 24 h (P < 0.05). The expression of Bcl-2 was reduced in the group after exposure to CHCS for 24 h (P < 0.05). (3).H.pylori was able to directly induce SGC-7901 cells apoptosis in dose-and time-dependent manner. The levels of △Ψm began to decrease after exposure to CHCS for 4 h. However, it decreased significantly after 8 h, and further to the lowest point at 12 h. The changes of Ca2 + concentration manifested a contrary manner. (4).The expression of COXⅠ,COXⅡand COX III decreased gradually after exposure to CHCS for 4 h. Especially after 8 h, the expression decreased significantly. When treated with CHCS after 12 h, the expression continued to decrease (P<0.05). The changes of COXⅠprotein showed the same trend. 3. Experiment in vivo: (1).The incidence rates of intestinal metaplasia and dysplasia in the H. pylori infected group were significantly higher than control (P < 0.05). (2).The apoptotic rates of H. pylori infected MGs were higher than those in the control group at the same period. Especially the apoptotic rate was the highest after H.pylori infected MGs for 4 week (P < 0.05). (3).The level of △Ψm at 4 week was dramatically lower than other groups (P < 0.05). The level of Ca2+ at 4 week was dramatically higher than the other groups (P < 0.05). There was no significant difference in the other groups after infected H. pylori for 12 and 24 week. (4).Immunohistochemistry showed weak positive staining of Bcl-2,Bax,Cyt-c proteins in the control group. Enhancement of expression was found in the group after H. pylori infection for 4, 12 and 24 week (P < 0.05). 4.Chemopreventive experiment in vivo and vitro: (1).At necropsy, in mice given celecoxib, the mean tumor weight were significantly lower than that of control group[(0.43±0.21 g) vs (1.33±0.45 g)],P<0.05.The tumor inhibition rate was 67.5%. (2).Celecoxib displayed a strong growth inhibition effect on SGC-7901 cells in a dose-and time-dependent manner. (3).After exposure to 50μmol/L celecoxib for 8 and 12 h, FCM analysis showed the G0/G1 phase ratio increased, S and G2/M phase ratio decreased in SGC-7901 cells.(4).The apoptotic rate was higher in the treatment group than in control group (P<0.05). (5).The levels of △Ψm began to decrease after treatment of celecoxib for 4 h.After 8h, it decreased distinctly. After 12 h, it continued to descend. After 24 h, it decreased to the lowest point. The changes of Ca2 + showed the contrary trend. (6).Increasing expression of cyclooxygenase-2 protein in the group after exposure to CHCS (P<0.05). Dramatically decreasing expression was found in the group after treatment of celecoxib.The expression of Caspase-9 protein elevated in all groups, especially in group exposure to CHCS and celecoxib(P<0.05). (7).RT-PCR results showed that cyclooxygenase-2 mRNA were elevated after exposure to CHCS (P<0.05). Decreasing expression of cyclooxygenase-2 mRNA were in the group after treated by celecoxib (P<0.05). CONCLUSIONS: 1.MGS models are the valuable to study the role of H. pylori in gastric carcinogenesis. H. pylori could direcly induce gastric carcinoma. 2.In the apoptotic process of H. pylori inducing SGC-7901 cells, it was observed that mitochondrial Ca2 +overloaded, △Ψm loss, Cyt-c released, the gene encoded by mitochondial changed , and the proteins decreased in the early stage of apoptosis.It suggested that H. pylori could induce mitochondrial structures and functions damage and make cells apoptosis through the mitochondrial pathway. 3.The apoptosis of gastric epithelial cells of MGs appeared in the early stage of H. pylori infection; mitochondrial △Ψm decreasing, Ca2 + increasing, MPT changing and Cyt-c releasing were taken part in the apoptotic process. It was modulated by mitochondrial membrane proteins Bcl-2 families. Mitochondrial damage may be one of the reasons which H. pylori induce the apoptosis of gastric epithelial cells. 4.Celecoxib could effectively inhibit the proliferation of gastric carcinoma, prevent precancerous lesions induced by H. pylori infection. The mechanisms may involve inhibiting cyclooxygenase-2 synthesis, inducing apoptosis of tumor cells and affecting various phases of cell cycle kinetics. Celecoxib has a better perspective in the chemoprevention and treatment of H. pylori related gastric carcinoma.