电针联合壮医药线点灸对糖尿病胃轻瘫大鼠胃窦组织凋亡相关蛋白表达的影响

Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis rats

目的:观察电针联合壮医药线点灸对糖尿病胃轻瘫(DGP)大鼠胃窦组织凋亡相关蛋白及基因表达的影响,探讨电针联合壮医药线点灸治疗DGP的作用机制。方法:SD大鼠随机分为正常组、模型组、西药组、电针组、点灸组、联合组,每组12只。采用链脲佐菌素腹腔注射法制备DGP大鼠模型。西药组予0.15 mg/mL枸橼酸莫沙必利混悬液灌胃给药;选取“中脘”“内关”“三阴交”穴,电针组电针20 min(10 Hz/50 Hz, 2 mA),点灸组每穴点灸3壮,联合组为电针联合点灸治疗。均每日1次,连续3周。检测各组大鼠随机血糖、胃排空率及小肠推进率;TUNEL法检测胃窦组织细胞凋亡指数;Western blot、荧光定量PCR法检测胃窦组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白及mRNA表达。结果:与正常组比较,模型组随机血糖升高(P<0.01),胃排空率、小肠推进率降低(P<0.01),胃窦组织可见大量凋亡细胞,细胞凋亡指数明显升高(P<0.01),Caspase-3、Bax蛋白及mRNA表达升高(P<0.01),Bcl-2蛋白及mRNA表达降低(P<0.01)。与模型组比较,电针组、点灸组、联合组随机血糖降低(P<0.01);4个治疗组胃排空率、小肠推进率均升高(P<0.01),胃窦组织细胞凋亡指数均降低(P<0.01);西药组、联合组Caspase-3、Bax蛋白及mRNA表达降低(P<0.01),Bcl-2蛋白及mRNA表达升高(P<0.01);点灸组Caspase-3蛋白及mRNA、Bax mRNA表达降低(P<0.01,P<0.05),Bcl-2 mRNA表达升高(P<0.01);电针组Caspase-3蛋白、Bax蛋白及mRNA表达降低(P<0.05,P<0.01),Bcl-2 mRNA表达升高(P<0.01)。与联合组比较,电针组随机血糖升高(P<0.01),胃排空率、小肠推进率降低(P<0.05,P<0.01),Caspase-3蛋白、Bax mRNA表达升高(P<0.01),Bcl-2蛋白及mRNA表达降低(P<0.05,P<0.01);点灸组随机血糖升高(P<0.05),胃排空率、小肠推进率降低(P<0.01),细胞凋亡指数升高(P<0.01),Caspase-3蛋白、Bax蛋白及mRNA表达升高(P<0.01,P<0.05),Bcl-2蛋白表达降低(P<0.05);西药组随机血糖升高(P<0.01),胃排空率、小肠推进率均降低(P<0.01)。电针组胃排空率、小肠推进率较西药组升高(P<0.05,P<0.01)。点灸组Bcl-2 mRNA表达较电针组升高(P<0.01)。结论:电针联合壮医药线点灸可在一定程度上降低DGP大鼠的血糖,提高胃肠动力,其作用机制可能与调节胃窦组织Caspase-3、Bax、Bcl-2蛋白及mRNA的表达,减少细胞凋亡有关。

Objective To observe the effect of electroacupuncture(EA) combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis(DGP) rats, so as to explore its mechanism underlying improvement of DGP. Methods Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion(moxibustion) and EA+moxibustion(combination) groups(12 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin(STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension(10 mL/kg). EA(10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion(3 cones) was applied to “Zhongwan”(CV12), bilateral “Neiguan”(PC6) and bilateral “Sanyinjiao”(SP6) of the related groups, once a day for 3 weeks. The blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The apoptosis index of gastric antrum cells were observed by TUNEL staining. The protein and mRNA expressions of Caspase-3, B-cell lymphoma/leukemia-2(Bcl-2) and Bcl-2 associated X protein(Bax) in gastric antrum were detected by Wes-tern blot and real-time quantitative PCR, respectively. Results Compared with the normal group, the blood glucose, the apoptosis index, the protein and gene expressions of Caspase-3 and Bax were significantly increased(P<0.01), and the gastric emptying rate, intestinal propulsive rate, the protein and gene expressions of Bcl-2 were considerably decreased(P<0.01) in the model group. In contrast to the model group, the blood glucose in the EA, moxibustion and combination groups, the apoptosis index in the 4 treatment groups, as well as Caspase-3 protein, Bax protein and mRNA expressions in the medication, EA and combination groups, Caspase-3 protein and mRNA, Bax mRNA expressions in the moxibustion group were significantly decreased(P<0.01, P<0.05);while the gastric emptying rate and intestinal propulsive rate in the 4 treatment groups, and Bcl-2 protein and mRNA expressions in the medication and combination groups, Bcl-2 mRNA expressions in the EA and moxibustion groups were obviously increased(P<0.01). The effects of EA+moxibustion were significantly superior to those of simple EA, moxibustion or medication in increasing gastric emptying rate and intestinal propulsive rate, and in lowering blood glucose(P<0.05, P<0.01). And the effects of the combination treatment were better than those of EA in lowering Caspase-3 protein and Bax mRNA expressions(P<0.01), and in increasing Bcl-2 protein and mRNA expressions(P<0.05, P<0.01). Also the effects of the combination treatment were better than those of moxibustion in lowering the apoptosis index, Caspase-3 protein, and Bax protein and mRNA expressions(P<0.01, P<0.05), and in increasing Bcl-2 protein expression(P<0.05). Conclusion EA combined with Zhuang-medicine-thread moxibustion can reduce blood glucose and improve gastrointestinal motility in DGP rats, which may be related to its effect in regulating of Caspase-3, Bax and Bcl-2 expression.