基于PI3K/Akt/mTOR通路探讨排石冲剂对草酸钙肾结石大鼠的作用机制

Action mechanism of Paishi Granule on calcium oxalate renal calculi in rats based on PI3K/Akt/mTOR pathway

目的探讨磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)通路在草酸钙结石形成中的作用机制及其在排石冲剂防治草酸钙肾结石中的影响。方法将40只雄性SPF级Wistar大鼠随机分为正常组、模型组、排石冲剂组和枸橼酸钾组,每组各10只。除正常组外,其他各组建立草酸钙结石模型。排石冲剂组给予排石冲剂药液,枸橼酸钾组给予枸橼酸钾药液。经4周干预后,检测各组大鼠血液和尿液中电解质、肌酐、尿素等生化指标,肾脏组织中超氧化物歧化酶(SOD)和丙二醛(MDA)水平。采用HE染色和VonKossa染色肾组织石蜡切片检测肾脏的损伤情况和结石的沉积情况。采用免疫组化法检测PI3K/Akt/mTOR通路相关蛋白表达水平。结果与模型组相比,排石冲剂组和枸橼酸钾组大鼠尿Ox、Ca^(2+)、P^(3+)明显降低(P<0.01),而尿Mg^(2+)则明显升高(P<0.01)。与模型组相比,排石冲剂组和枸橼酸钾组大鼠血肌酐(Cr)、尿素氮(BUN)、Ca^(2+)水平明显下降(P<0.01,<0.05),排石冲剂组大鼠P^(3+)水平下降(P<0.05),而血Mg^(2+)则升高(P<0.05)。与模型组相比,排石冲剂组和枸橼酸钾组大鼠肾组织中SOD明显上升(P<0.01),MDA明显下降(P<0.01),与模型组相比,排石冲剂组大鼠肾小管扩张明显减轻,同时排石冲剂组、枸橼酸钾组肾小管黑色结晶沉积明显减少。与模型组比较,排石冲剂组和枸橼酸钾组大鼠肾组织中p-PI3K、Akt的IOD值明显下降(P<0.01),排石冲剂组大鼠肾组织中mTOR的IOD值下降(P<0.05);PI3K在各组表达基本一致。结论排石冲剂防治肾结石的作用机制,可能是通过减少氧化应激,继而抑制PI3K/Akt/mTOR通路,以降低草酸钙的沉积,减少肾小管的损伤。

Objective To investigate the mechanism of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway in calcium oxalate renal calculus and its effects on Paishi Granule in the prevention and treatment of calcium oxalate renal calculus.Methods Fourty male Wistar rats were randomly divided into normal group,model group,Paishi Granule group and potassium citrate group to establish calcium oxalate renal calculus model by ethylene glycol and ammonium chloride,10 in each.Rats were sacrificed after 4 weeks of intervention.Physiological data such as body weight and urine volume,biochemical indexes such as electrolyte,creatinine,and urea,as well as superoxide dismutase(SOD)and malondialdehyde(MDA)levels and PI3K/Akt/mTOR pathway related protein expression levels were detected.Results Compared with model group,urinary Ox,Ca^(2+),and P^(3+)were significantly lower(P<0.01),while urinary Mg^(2+)was significantly higher(P<0.01)in lithotripsy flush group and potassium citrate group.Compared with model group,the levels of Cr,BUN,Ca^(2+)were significantly decreased in lithotripsy flush group and potassium citrate group(P<0.05,<0.01),and the levels of P^(3+)were decreased in lithotripsy flush group(P<0.05),while blood Mg^(2+)was increased in lithotripsy flush group(P<0.05).Compared with model group,SOD was significantly increased(P<0.01)and MDA was significantly decreased(P<0.01)in the kidney tissues of rats in lithotripter and potassium citrate groups,indicating that lithotripter and potassium citrate had antioxidative stress effects.Compared with model group,the dilatation of renal tubules was significantly reduced in lithotripsy flush group,while the black crystalline deposits in the renal tubules were significantly reduced in lithotripsy flush and potassium citrate groups.Compared with model group,the IOD values of p-PI3K and Akt in the kidney tissues of rats in lithotripter and potassium citrate groups were significantly decreased(P<0.01),and the IOD values of m TOR in the kidney tissues of rats in lithotripter group were decreased(P<0.05);the expression of PI3K was basically the same in all groups.Conclusion The mechanism of action of lithotripsy punch against kidney stones may be through reducing oxidative stress and subsequently inhibiting PI3K/Akt/mTOR pathway to reduce calcium oxalate deposition and tubular damage.