健脾祛湿方对脾虚湿盛型高脂血症大鼠血脂、胃肠功能、水液代谢的影响及作用机制

Effect and Mechanism of Jianpi Qushi Prescription on Blood Lipids, Gastrointestinal Function and Water Metabolism of Hyperlipidemic Rats with Spleen Deficiency and Dampness

目的:观察健脾祛湿方对脾虚湿盛型高脂血症大鼠血脂、胃肠功能、水液代谢的影响及作用机制。方法:将56只SD大鼠随机分为空白组(n=8)和造模组(n=48),采用“劳倦过度+饮食不节+高脂饲料喂养”复制脾虚湿盛型高脂血症大鼠模型。造模4周后,根据总胆固醇(TC)水平将造模组随机分成6组:即模型组,血脂康组,参苓白术颗粒组,健脾祛湿方低、中、高剂量组,每组8只。分组后开始给药,灌胃剂量为1 mL/100 g,空白组、模型组给予生理盐水,其余组给予相应的受试物,连续给药6周。测定血脂四项TC、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-Ch)和高密度脂蛋白胆固醇(HDL-Ch),胃肠激素,即胃动素(MTL)、促胃液素(GAS)和血管活性肠肽(VIP),水液调节激素醛固酮(ALD)、抗利尿激素(ADH)和心房利尿钠肽(ANP),以及白蛋白(ALB)和总蛋白(TP),采用苏木精-伊红(HE)染色法观察胃、结肠组织形态,免疫荧光法检测结肠水孔蛋白(AQP)3、胃AQP4的表达位置及水平,采用蛋白质印迹法(Western Blotting)检测结肠、胃组织中闭合蛋白(Occludin)的表达量。结果:与正常组比较,模型组大鼠血清TC、TG、LDL-Ch水平升高(P<0.001),HDL-Ch水平降低(P<0.05),血清MTL、GAS水平降低(P<0.001,P<0.01),VIP升高(P<0.001),ALD、ADH升高(P<0.05,P<0.001),ANP水平显著降低(P<0.001),TP、ALB水平降低(P<0.001,P<0.05)。结肠绒毛、胃黏膜出现大量脱落情况,结肠AQP3荧光强度降低,胃AQP4荧光表达增强,结肠、胃组织Occludin表达水平降低。与模型组比较,血脂康组、参苓白术颗粒组、健脾祛湿方低、中、高剂量组大鼠血清TC、TG、LDL-Ch水平呈下降趋势,HDL-C水平升高,MTL、GAS显著升高,VIP水平显著降低,ALD、ADH水平降低,ANP显著升高,结肠、胃组织形态结构有所改善。结肠AQP3荧光表达增强,胃AQP4荧光强度减弱,结肠、胃组织Occludin表达水平增强。结论:健脾祛湿方可降低脾虚湿盛型高脂血症大鼠血脂,有健脾祛湿之功,其作用机制可能是通过调节Occludin、AQP3、AQP4的表达,保护紧密连接结构的完整性达到促进胃肠消化吸收功能,改善水液代谢障碍的作用。

Objective:To observe the effect and mechanism of Jianpi Qushi Prescription(JPQS) on blood lipids, gastrointestinal function and water metabolism of hyperlipidemic rats with spleen deficiency and dampness.Methods:A total of 56 SD rats were randomly divided into blank group(n=8) and modeling group(n=48).A hyperlipidemic rat model with spleen deficiency and dampness was replicated by overfatigue+irregular diet+high-fat feeding.After 4 weeks of modeling, the modeling group was randomly divided into 6 groups according to total cholesterol(TC) level: model group, Xuezhikang group, Shenling Baizhu Granules group, and JPQS low-,medium-and high-dose groups, with 8 in each group.After grouping, drugs were administered by gavage at a dose of 1 mL/100 g.The blank group and the model group were given normal saline, while the other groups were given corresponding drugs for 6 consecutive weeks.Four blood lipid indexes[TC,triglycerides(TG),low-density lipoprotein cholesterol(LDL-Ch),high-density lipoprotein cholesterol(HDL-Ch)],gastrointestinal hormones[motilin(MTL),gastrin(GAS),vasoactive intestinal peptide(VIP)],water regulation hormones[aldosterone(ALD),anti-diuretic hormone(ADH),atrial natriuretic hormone(ANP)],albumin(ALB) and total protein(TP) were detected.Hematoxylin-eosin(HE) staining was used to observe the morphology of gastric and colonic tissues.Immunofluorescence assay was performed to detect the expression position and level of aquaporin 3(AQP3) in colon and AQP4 in stomach, and Western blot was performed to detect the expression level of Occludin in colonic and gastric tissues.Results:Compared with the blank group, the model group had increased serum levels of TC,TG,and LDL-Ch(P<0.001),VIP(P<0.001) and ALD and ADH(P<0.05,P<0.001),and decreased serum levels of HDL-Ch(P<0.05),MTL and GAS(P<0.001,P<0.01),ANP(P<0.001) and TP and ALB(P<0.001,P<0.05).Additionally, colon villi and gastric mucosa were shed in large amounts in the model group, with reduced fluorescence intensity of AQP3 in colon, enhanced fluorescence expression of AQP4 in stomach, and down-regulated expression of Occludin in colon and gastric tissues.Compared with the conditions in the model group, the serum levels of TC,TG and LDL-Ch of rats in administration groups presented a downward trend, while the HDL-C level was elevated;MTL and GAS were significantly up-regulated, while VIP was significantly reduced;a decrease was found in ALD and ADH,while a remarkable increase was found in ANP;the morphological structure of colonic and gastric tissues was improved, with enhanced fluorescence expression of AQP3 in colon, weakened fluorescence intensity of AQP4 in stomach and elevated expression of Occludin in colonic and gastric tissues.Conclusion:JPQS reduces the blood lipids of hyperlipidemic rats with spleen deficiency and dampness and shows the effect of invigorating spleen and removing dampness.The mechanism may be regulating the expressions of Occludin, AQP3 and AQP4 to protect the structural integrity of tight junctions and promote gastrointestinal digestion and absorption, thereby improving the water metabolism disorders.