Nesfatin-1对离体胃黏膜细胞胃酸分泌的影响

目的:研究nesfatin-1对离体培养的大鼠胃黏膜酸分泌的影响,探讨nesfatin-1对H+/K+-ATP酶mRNA及蛋白表达的影响.方法:酶解法分离大鼠胃黏膜细胞,细胞免疫荧光检测法鉴定细胞.用不同浓度的nesfatin-1(10-4-10-1μmol/L)对胃黏膜细胞进行处理0、1、2、3、4h,设立空白对照组,以14C-氨基比林摄取为酸分泌指标,检测nesfatin-1对大鼠离体的胃黏膜细胞酸分泌的影响.用RT-PCR法及Western印迹法检测nesfatin-1对胃黏膜细胞H+/K+-ATP酶alpha(α)亚基和beta(β)亚基mRNA及蛋白表达的影响.结果:Nesfatin-1在10-1μmol/L浓度下,在1、2h能够抑制离体培养的大鼠胃黏膜细胞的酸分泌.Nesfatin-1(10-1μmol/L)在1、2、3h均能够抑制H+/K+-ATP酶α亚基mRNA表达水平;在1、2h能够抑制H+/K+-ATP酶β亚基mRNA表达水平,分别与对照组相比,差异有统计学意义(均P<0.01).Nesfatin-1(10-4-10-1μmol/L)作用胃黏膜细胞2h时,呈剂量依赖性抑制α亚基和β亚基mRNA表达水平,分别与对照组相比,差异有统计学意义(均P<0.01).Nesfatin-1(10-1μmol/L)在1、2、3h能够抑制α亚基蛋白表达水平;在2、3h能够抑制β亚基蛋白表达水平,分别与对照组相比,差异有统计学意义(均P<0.01).Nesfatin-1(10-3-10-1μmol/L)作用胃黏膜细胞2h时,呈剂量依赖性抑制α亚基和β亚基蛋白表达水平,与对照组相比,差异有统计学意义(均P<0.01).结论:Nesfatin-1能抑制离体培养的大鼠胃黏膜细胞的酸分泌,有可能是通过下调H+/K+-ATP酶α亚基和β亚基的mRNA及蛋白表达的水平影响酸分泌.

Nesfatin-1 inhibits gastric acid secretion by cultured rat gastric mucosa cells

AIM:To clarify the effect of nesfatin-1 on gastric acid secretion and the expression of the H+/K+ATPase mRNA and protein in rat gastric mucosa cells in vitro.METHODS:Gastric mucosa cells were isolated from SD rats by enzymolysis and identified by immunofluorescence staining.Cultured rat gastric mucosa cells were divided into control group and nesfatin-1 group,and the nesfatin-1 group was pretreated with different concentrations(0,10-4,10-3,10-2,10-1 μmol/L) of nesfatin-1 for different durations(0,1,2,3,4 h).The effect of nesfatin-1 on gastric acid secretion was investigated by monitoring 14C-aminopyrine(14C-AP) accumulation,and the expression of H+/K+ATPase α and β subunit mRNA and protein wasexamined by real-time PCR and Western blot.RESULTS:Pretreatment with nesfatin-1 at a dose of 10-1 or 10-2 μmol/L for 2 or 3 h inhibited gastric acid secretion,but nesfatin-1 at a dose of 10-3 or 10-4 μmol/L had no such effect.Nesfatin-1 at a dose of 10-1 μmol/L inhibited the expression of H+/K+-ATPase α subunit mRNA after pretreatment for 1,2,or 3 h and inhibited the expression of H+/K+-ATPase β subunit mRNAs after pretreatment for 1 or 2 h.In the dose range between 10-4 to 10-1 μmol/L,nesfatin-1 dose-dependently inhibited the expression of H+/K+-ATPase α subunit and β subunit mRNA after pretreatment for 2 h.Nesfatin-1 at a dose of 10-1 μmmol/L inhibited H+/K+-ATPase α subunit protein expression after pretreatment for 1,2 or 3 h and inhibited H+/K+-ATPase β subunit protein expression after pretreatment for 2 or 3 h.In the dose range between 10-3 to 10-1 μmol/L,nesfatin-1 dosedependently inhibited H+/K+-ATPase α and β subunit protein expression after pretreatment for 2 h.CONCLUSION:Our data suggest that nesfatin-1 inhibits gastric acid secretion by rat gastric mucosa cells in vitro possibly by down-regulating the expression of H+/K+-ATPase mRNA and protein.