青蒿琥酯调节PI_(3)K/Akt信号通路对肾草酸结石形成的影响

Impacts of Artesunate on the Formation of Kidney Oxalate Stones by Regulating the PI_(3)K/Akt Signaling Pathway

目的探讨青蒿琥酯(artesunate,Art)对肾结石形成及磷脂酰肌醇-3激酶(phosphatidylinositol 3 kinase,PI_(3)K)/蛋白激酶B(protein kinase B,Akt)信号通路的影响。方法实验分为对照组(Control组)、模型组(Model组)、青蒿琥酯低剂量组(Art-L组)、青蒿琥酯高剂量组(Art-H组)、青蒿琥酯高剂量+PI_(3)K抑制剂组(Art+LY294002组)。通过乙二醇饮水和草酸钠腹腔注射建立肾结石大鼠模型;HK-2细胞暴露于200mg/L一水草酸钙晶体和2mmol/L草酸盐中建立肾结石细胞模型。检测尿液Ca^(2+)、草酸(oxalic acid,Ox)及血液血清肌酐(serum creatinine,Scr)、血尿素氮(blood urea nitrogen,BUN)水平;Pizzolato染色测量肾组织钙盐沉积;HE染色测定肾病理形态;丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)试剂盒检测肾组织和HK-2细胞MDA、SOD水平;ELISA法检测肾组织和HK-2细胞TNF-α、IL-1β和IL-6水平;荧光定量检测Bax、caspase-3及PI_(3)K、Akt水平;免疫组化和Western blot法测量PI_(3)K、Akt蛋白表达;流式细胞术测量HK-2细胞凋亡。结果与Model组比较,Art-L组、Art-H组Ca^(2+)、Ox、Scr、BUN、肾组织和HK-2细胞MDA、TNF-α、IL-1β、IL-6及肾组织Bax、caspase-3下降,肾组织和HK-2细胞SOD、PI_(3)K、Akt升高(P<0.05);与Art-H组比较,Art+LY294002组Ca^(2+)、Ox、Scr、BUN、肾组织和HK-2细胞MDA、TNF-α、IL-1β、IL-6及肾组织Bax、caspase-3升高,肾组织和HK-2细胞SOD、PI_(3)K、Akt下降(P<0.05)。结论青蒿琥酯可以可以抑制肾结石的形成;其抑制结石形成可能是通过上调PI_(3)K/Akt信号通路的机制产生的。

Objective To investigate the impacts of artesunate(Art)on the formation of kidney stones and the phosphatidylinositol 3 kinase(PI_(3)K)/protein kinase B(Akt)signaling pathway.Methods Rats were randomly divided into the Control group,Model group,Artesunate low-dose group(ART-L group),artesunate high-dose group(ART-H group),artesunate high-dose+PI_(3)K inhibitor group(Art+LY294002 group).The rat model of kidney stone was established by drinking water with ethylene glycol and injecting sodium oxalate intraperitoneally.A kidney stone cell model was established by exposing HK-2 cells to 200 mg/L calcium oxalate monohydrate crystals and 2 mmol/L oxalate.The levels of Ca^(2+)and oxalic acid(Ox)in urine and serum creatinine(Scr)and blood urea nitrogen(BUN)were detected;Pizzolato staining was applied to observe the deposition of calcium salts in renal tissues;the pathological morphology in renal tissues was detected by HE staining;malondialdehyde(MDA)and superoxide dismutase(SOD)kits were used to detect the levels of MDA and SOD in renal tissue and HK-2 cells;ELISA method was applied to detect TNF-α,IL-1βand IL-6 in renal tissue and HK-2 cells;fluorescence quantification was applied to detect the levels of Bax,caspase-3,PI_(3)K and Akt;immunohistochemical and Western blot were applied to detect the expression levels of PI_(3)K and Akt proteins in the renal tissues.HK-2 cell apoptosis was measured by flow cytometry.Results Compared with the Model group,Ca^(2+),Ox,Scr,BUN,the level of MDA,TNF-α,IL-1β,IL-6 in renal tissue and HK-2 cells,the expression of Bax,caspase-3 in renal tissue decreased in Art-L and Art-H groups,the level of SOD,PI_(3)K,and Akt in renal tissue and HK-2 cells increased(P<0.05);compared with the Art-H group,Ca^(2+),Ox,Scr,BUN,the level of MDA,TNF-α,IL-1β,IL-6 in renal tissue and HK-2 cells,the expression of Bax,caspase-3 in renal tissue increased in the Art+LY294002 group,the level of SOD,PI_(3)K and Akt in renal tissue and HK-2 cells decreased(P<0.05).Conclusion Artesunate can inhibit the formation of kidney stones;its inhibition of stone formation may be produced through the mechanism of up-regulating the PI_(3)K/Akt signaling pathway.