硫化氢合成酶在糖尿病模型小鼠结肠动力障碍中的作用机制

The role of hydrogen sulfide synthesis in colonic dysmotility of diabetic mice and the underlying mechanism

目的探讨硫化氢(H_(2)S)合成酶在链脲佐菌素(STZ)诱导的雄性C57BL/6糖尿病模型小鼠结肠动力障碍中的作用。方法将16只SPF级小鼠随机分为STZ组和对照组,每组各8只。再将10只SPF级小鼠随机分为正常组和河豚毒素(TTX)组,每组各5只。STZ组一次性腹腔注射STZ(200 mg/kg)诱导1型糖尿病模型,对照组腹腔注射等体积枸橼酸缓冲液。正常组和TTX组不做任何处理。采用免疫荧光检测H_(2)S合成酶[胱硫醚-γ-裂解酶(CSE)和胱硫醚-β-合成酶(CBS)]在STZ组和对照组小鼠结肠的分布;采用Western blot检测两组小鼠结肠组织中CBS和CSE的表达水平;通过恒温浴槽实验观察4组小鼠结肠平滑肌收缩改变;通过膜片钳实验观察不同浓度外源性H_(2)S对正常组小鼠平滑肌细胞L型钙通道电流的影响。结果STZ组小鼠STZ注射后第2周与第4周空腹血糖水平均显著高于同期对照组,排便粒数均显著低于同组注射前及同期对照组(P<0.001)。STZ组小鼠LMS和CMS收缩振幅均低于对照组(P<0.05)。STZ组小鼠结肠组织CBS和CSE表达水平均高于对照组(P<0.05)。低浓度H_(2)S供体硫氢化钠(NaHS,0.10 mmol/L)促进小鼠结肠平滑肌收缩,而高浓度NaHS(0.50 mmol/L~1.50 mmol/L)抑制其收缩,且TTX不能阻断NaHS对肌条的双向调节作用。正常小鼠NaHS(0.10 mmol/L)组电流密度高于NaHS(0.50 mmol/L)组及基线值(P<0.05)。结论糖尿病模型小鼠结肠动力障碍可能与肠道组织H_(2)S合成增加进而抑制平滑肌细胞L型钙通道开放有关。

Objective To investigate the role of hydrogen sulfide(H_(2)S)synthesis in colonic dysmotility of male C57 BL/6 diabetic mice induced by streptozotocin(STZ).Methods 16 SPF mice were randomly divided into STZ group and control group with 8 mice in each group.10 SPF mice were randomly divided into normal group and tetrodotoxin(TTX)group with 5 mice in each group.The type 1 diabetes model(T1 DM)was induced by a single intraperitoneal injection of STZ(200 mg/kg).Control mice were injected with the same volume of citrate buffer.Mice in normal group and TTX group did not do any treatment.Immunohistochemistry was applied to observe the distribution of H_(2)S-producing enzymes cystathionine-γ-lyase(CSE)and cystathionine-β-synthase(CBS).Western blot were performed to evaluate the expression levels of CBS and CSE in the colon.Organ bath system was used to observe the spontaneous contraction of colonic smooth muscle.Patch clamp technique was applied to record the currents of L-type calcium channel in smooth muscle cells.Results The fasting blood glucose levels of mice in STZ group at the 2 nd and the 4 th week after STZ injection were significantly higher than those of the control group,and the number of fecal pellets were significantly lower than those before injection in the same group and those of the control group(P<0.001).The contraction amplitude of LMS and CMS of colon in STZ group was lower than that in control group(P<0.05).The expression levels of CBS and CSE in colon tissue of STZ group mice were higher than those of control group(P<0.05).Sodium hydrosulfide(NaHS),the donor of exogenous H_(2)S,exerted an excitatory effect on the colonic muscle contraction at lower concentration(0.10 mmol/L)while inhibited the muscle contraction at higher concentrations(0.50 mmol/L-1.50 mmol/L).And TTX failed to block the dual effects of NaHS.The current density of NaHS(0.10 mmol/L)group in the normal mice was higher than those in NaHS(0.50 mmol/L)group and the baseline values.Conclusion The dysfunction of colonic motility in diabetic mice may be related to the increased synthesis of H_(2)S in colon with subsequent inhibition of the opening of L-type calcium channels in smooth muscle cells.