摘要:
目的 探讨富氢水对乙醇诱导急性胃损伤的保护作用及其机制.方法 采用实验研究方法.40只昆明小鼠按随机数字表法分为正常对照组(0.01 mL/g生理盐水+0.03 mL/g生理盐水)、单独富氢水组(0.01 mL/g生理盐水+0.03 mL/g饮用富氢水)、乙醇造模组(0.01mL/g 56.酒精饮品+0.03 mL/g生理盐水)和富氢水治疗组(0.01 mL/g 56.酒精饮品+0.03 mL/g饮用富氢水),每组10只小鼠.每天灌胃2次,连续灌胃7d.检测指标:(1)胃溃疡指数测定.(2)胃组织病理学检查.(3)检测血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的表达水平.(4)检测胃组织中SOD和MDA的表达水平.(5)免疫组织化学染色检测小鼠胃组织中凋亡因子caspase-3、bax、caspase-9、fas和caspase-8的蛋白表达水平.(6)逆转录-聚合酶链反应(RT-PCR)检测小鼠胃组织中凋亡因子caspase-3、bax、caspase-9、fas和caspase-8的mRNA相对表达水平.正态分布的计量资料以(x)±s表示,多组间比较采用单因素方差分析,两组间比较采用LSD-t检验.结果 (1)胃溃疡指数测定:正常对照组、单独富氢水组、乙醇造模组、富氢水治疗组小鼠胃黏膜损伤指数分别为0、0、10.40± 1.64、3.92±0.23,4组比较,差异有统计学意义(F=175.050,P<0.05);乙醇造模组分别与正常对照组和富氢水治疗组比较,差异均有统计学意义(t=19.835,12.352,P<0.05).(2)胃组织病理学检查:①胃组织大体标本:正常对照组及单独富氢水组小鼠胃黏膜完整光滑,未见糜烂、溃疡及炎症.乙醇造模组小鼠部分胃黏膜糜烂、溃疡损伤面积大,程度重;与乙醇造模组小鼠比较,富氢水治疗组小鼠胃黏膜损伤面积减少.②胃组织病理学检查结果显示:正常对照组及单独富氢水组小鼠胃黏膜表面完整.与正常对照组比较,乙醇造模组小鼠部分胃黏膜表面上皮变性受损.富氢水治疗组小鼠与乙醇造模组小鼠比较,胃黏膜坏死变性程度和炎性细胞浸润程度均减轻.(3)血清中SOD、MDA、IL-6、TNF-α的表达水平:正常对照组、单独富氢水组、乙醇造模组、富氢水治疗组小鼠血清SOD分别为(70±6) U/mL、(74±4)U/mL、(101±4)U/mL、(115±5)U/mL,MDA分别为(7.52±0.23)μmol/L、(7.61±0.91)μmol/L、(16.95±0.66) μmoL/L、(14.02±0.58) μmoL/L,SOD/MDA分别为9.40±1.07、9.91±1.55、5.99±0.17、8.23±0.32,IL-6分别为(6.3±1.8) ng/L、(5.1±1.6) ng/L、(19.2±4.9) ng/L、(7.1±1.8) ng/L,4组上述指标比较,差异均有统计学意义(F=97.405,269.950,16.486,25.663,P<0.05);TNF-α分别为(53±14) ng/L、(67±17)ng/L、(52±13) ng/L、(58±21) ng/L,4组比较,差异无统计学意义(F=0.862,P>0.05).(4)胃组织中SOD和MDA的表达水平:正常对照组、单独富氢水组、乙醇造模组、富氢水治疗组小鼠胃组织中SOD分别为(93±18) U/mL、(93±13) U/mL、(121±31) U/mL、(143±26)U/mL,MDA分别为(7.90±1.72) μmol/L、(6.96±1.49) μmol/L、(17.10±4.88) μmol/L、(7.31±1.58) μmol/L,SOD/MDA分别为12.48±4.54、13.83±3.40、7.88±3.70、20.00±4.68,4组上述指标比较,差异均有统计学意义(F=5.463,15.051,7.388,P<0.05).(5)胃组织中凋亡相关蛋白的表达情况:免疫组织化学染色检测结果显示:正常对照组、单独富氢水组、乙醇造模组、富氢水治疗组小鼠胃组织中caspase-3蛋白表达水平分别为0.065 5±0.003 7、0.065 7±0.002 7、0.330 7±0.017 3、0.096 7±0.003 0,bax蛋白表达水平分别为0.065 7±0.003 0、0.064 9±0.003 0、0.335 4±0.033 3、0.084 8±0.001 7,fas蛋白表达水平分别为0.225 4±0.024 3、0.246 0±0.022 3、0.397 0±0.028 5、0.375 0±0.035 6,4组上述指标比较,差异均有统计学意义(F=1 004.222,309.171,48.555,P<0.05);caspase-9蛋白表达水平分别为0.049 2±0.0004、0.047 9±0.002 0、0.047 0±0.003 7、0.048 7±0.0008,caspase-8蛋白表达水平分别为0.151 5±0.010 2、0.154 0±0.013 5、0.157 2±0.006 2、0.153 9±0.006 3,4组上述指标比较,差异均无统计学意义(F=0.998,0.297,P>0.05).(6)胃组织凋亡相关因子mRNA的表达情况:RT-PCR检测结果显示:正常对照组、单独富氢水组、乙醇造模组、富氢水治疗组小鼠胃组织中caspase-3 mRNA相对表达水平分别为1.00±0.00、0.72±0.43、3.19±0.87、0.49±0.16,bax mRNA表达水平分别为1.00±0.00、0.66±0.26、1.58±0.76、0.69±0.25,caspase-9 mRNA相对表达水平分别为1.00±0.00、1.57±0.31、3.04±1.15、2.98±0.85,fas mRNA相对表达水平分别为1.00±0.00、0.50±0.19、2.84±0.98、0.53±0.24,4组上述指标比较,差异均有统计学意义(F=32.106,5.038,9.706,23.387,P<0.05);caspase-8 mRNA相对表达水平分别为1.00±0.00、1.50±0.60、1.36±0.34、1.32±0.43,4组比较,差异无统计学意义(F=1.337,P>0.05).结论 富氢水可以减轻乙醇诱导的急性胃损伤,其可能作用机制为抗氧化、抗炎和抗凋亡.富氢水安全可靠,未发现对机体存在毒副作用.%Objective To explore the protective effect and mechanism of hydrogen-rich water (HRW) on ethanol-induced acute gastric injury.Methods The experimental study was conducted.Forty kunming mice were divided into the 4 groups by random number table method:normal control group [0.01 mL/g normal saline (NS)+ 0.03 mL/g NS],HRW group (0.01 mL/g NS +0.03 mL/g HRW),ethanol model group (0.01 mL/g 56°alcoholic drinks +0.03 mL/g NS),HRW treated group (0.01 mL/g 56°alcoholic drinks +0.03 mL/g HRW).Ten mice in each group were administrated twice a day for 7 days.Testing indicators:(1) gastric ulcer index was measured,(2) pathological examination of gastric tissues,(3) activity of serum superoxide dismutase (SOD) and expressions of malondialdehyde (MDA),interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were measured,(4) expressions of SOD and MDA in gastric tissues were measured,(5) protein expressions of apoptosis related factors (caspase-3,bax,caspase-9,fas and caspase-8) in gastric tissues were measured by immunohistochemistry,(6) relative expressions of mRNA of apoptosis related factors (caspase-3,bax,caspase-9,fas and caspase-8) in gastric tissues were measured by realtime polymerase chain reaction (RT-PCR).Measurement data with normal distriburion were presented as (x)±s.Comparisons among groups were done using the one-way ANOVA and comparison between groups was done using the LSD-t test.Results (1) Gastric ulcer index was measured:gastric ulcer index of mice in the normal control group,HRW group,ethanol model group and HRW treated group were respectively 0,0,10.40± 1.64 and 3.92 ± 0.23,with statistically significant differences (F=175.050,P<0.05).There was a statistically significant difference between the ethanol model group and normal control group or HRW treated group (t =19.835,12.352,P< 0.05).(2) Pathological examination pathological examination of gastric tissues:① Macropathology of gastric tissues:the surface of the gastric mucosa was normal and smooth in the normal control group and the HRW group,without ulcer,erosion and inflammation.The partial gastric mucosal erosion and ulcer in the ethanol model group was large and very severe.Compared with the ethanol model group,the area of gastric mucosal lesion was reduced in the HRW treated group.② Results of pathological examination of gastric tissues:gastric mucosa in the normal control group and HRW group were integrity.Compared with the normal control group,the partial gastric surface epithelium was degenerate and impaired in the ethanol model group.Compared with the ethanol model group,the gastric mucosal erosion and inflammatory cell infiltration were alleviated in the HRW treated group.(3) Expressions of serum SOD,MDA,IL-6 and TNF-α:expressions of serum SOD,MDA,SOD/MDA and IL-6 were respectively (70±6)U/mL,(7.52±0.23) μmol/L,9.40 ± 1.07,(6.3 ± 1.8) ng/L in the normal control group and (74 ± 4) U/mL,(7.61 ±0.91) μmol/L,9.91 ± 1.55,(5.1 ± 1.6)ng/ L in the HRW group and (101 ± 4) U/mL,(16.95 ± 0.66) μmol/L,5.99±0.17,(19.2±4.9) ng/L in the ethanol model group and (115±5) U/mL,(14.02±0.58) μmol/L,8.23±0.32,(7.1±1.8)ng/L in the HRW treated group,with statistically significant differences among the 4 groups (F=97.405,269.950,16.486,25.663,P<0.05).The serum TNF-α levels were respectively (53± 14) ng/L,(67± 17) ng/L,(52± 13) ng/L,(58±21) ng/L in the above 4 groups,with no significant difference (F=0.862,P>0.05).(4) Expressions of SOD and MDA in gastric tissues were measured:expressions of SOD and MDA and SOD/MDA were respectively (93 ± 18) U/mL,(7.90± 1.72) μmol/L,12.48±4.54 in the normal control group and (93±13) U/mL,(6.96± 1.49) μmol/L,13.83±3.40 in the HRW group and (121±31) U/mL,(17.10±4.88) μmoL/L,7.88± 3.70 in the ethanol model group and (143 ± 26) U/mL,(7.31 ± 1.58) μmoL/L,20.00±4.68 in the HRW treated group,with statistically significant differences among the 4 groups (F=5.463,15.051,7.388,P< 0.05).(5) The expressions of apoptosis related factors in gastric tissues:the results of immunohistochemistry showed that the levels of caspase-3,bax and fas were repectively 0.065 5± 0.003 7,0.065 7±0.003 0,0.225 4±0.024 3 in the normal control group and 0.065 7±0.002 7,0.064 9±0.003 0,0.246 0±0.022 3 in the HRW group and 0.330 7±0.017 3,0.335 4±0.033 3,0.397 0±0.028 5 in the ethanol model group and 0.096 7±0.003 0,0.084 8±0.001 7,0.375 0±0.035 6 in the HRW treated group,showing statistically significant differences among the 4 groups (F=1 004.222,309.171,48.555,P<0.05).The levels of caspase-9 and caspase-8 were respectively 0.049 2±0.000 4,0.151 5±0.010 2 in the normal control group and 0.047 9±0.002 0,0.154 00.013 5 in the HRW group and 0.047 0±0.003 7,0.157 2±0.006 2 in the ethanol model group and 0.048 7±0.000 8,0.153 9±0.006 3 in the HRW treated group,with no statistically significant difference among the 4 groups (F=0.998,0.297,P>0.05).(6) The mRNA expressions of apoptosis related factors in gastric tissues:resutls of RT-PCR showed that relative expressions of mRNA of caspase-3,bax,caspase-9 and fas were respectively 1.00±0.00,1.00±0.00,1.00±0.00,1.00±0.00 in the normal control group and 0.72±0.43,0.66±0.26,1.57±0.31,0.50±0.19 in the HRW group and 3.19±0.87,1.58±0.76,3.04± 1.15,2.84±0.98 in the ethanol model group and 0.49±0.16,0.69±0.25,2.98±0.85,0.53±0.24 in the HRW treated group,showing statistically significant differences among the 4 groups (F=32.106,5.038,9.706,23.387,P<0.05).The mRNA levels of caspase-8 were respectively 1.00±0.00,1.50±0.60,1.36±0.34,1.32±0.43 in normal control group,HRW group,ethanol model group and HRW treated group,with no significant difference among the 4 groups (F=1.337,P>0.05).Conclusions Hydrogen-rich water could alleviate ethanolinduced acute gastric injury by antioxidant,anti-inflammatory and anti-apoptosis.Hydrogen-rich water is safe and reliable,without toxic and side effects on the body.