番石榴叶总三萜对脓毒症大鼠急性肾损伤的治疗及对PI3K/AKT通路的调控

Therapeutic effects of total triterpenoids in guava leaves on acute kidney injury in septic rats and its regulation of PI3K/AKT pathway

目的探讨番石榴叶总三萜对脓毒症大鼠急性肾损伤的治疗作用,并研究其对磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)通路的调控机制。方法自60只SD大鼠中随机选取50只建立脓毒症模型,余10只记为BC组,将建模大鼠随机分为MC组、PC组、LD组、MD组和HD组,PC组予以20万U·kg^(-1)·d^(-1)乌司他丁腹腔注射,LD组、MD组和HD组予以60、120和240 mg·kg^(-1)·d^(-1)番石榴叶总三萜腹腔注射,连续1周。酶联免疫吸附试验(ELISA)检测给药前后血清肌酐(Scr)、血尿素氮(BUN)水平;ELISA检测给药前后血清白介素-1β(IL-1β)、白介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)水平;苏木素-伊红染色(HE)观察肾组织病理改变;实时荧光定量聚合酶链反应(RT-PCR)检测PI3K、AKT、核因子-2-相关因子-2(Nrf2)mRNA表达;蛋白质免疫印迹法(WB)检测PI3K、AKT、Nrf2蛋白表达及磷酸化PI3K(p-PI3K)、磷酸化AKT(p-AKT)水平。结果给药后BC组血清Scr、BUN、IL-1β、IL-18、TNF-α水平均无明显变化(P>0.05),MC组均较给药前显著升高(P<0.01),其余4组均较给药前显著下降(P<0.01),且给药前MC组、PC组和3剂量组均高于BC组(P<0.01),给药后MC组均显著高于BC组(P<0.05),PC组和3剂量组均显著低于MC组(P<0.01),PC组、MD组和HD组均显著低于LD组(P<0.01),HD组均显著低于PC组、MD组(P<0.01);MC组肾组织病理严重改变,PC组和3剂量组均有所减轻,HD组肾组织病理学观察结果与BC组最为接近;各组肾组织PI3K、AKT mRNA及蛋白相对表达量比较差异均无统计学意义(P>0.05);4组肾组织Nrf2 mRNA及蛋白相对表达量、p-PI3K、p-AKT水平对比,MC组均显著高于BC组(P<0.01),PC组和3剂量组均显著低于MC组(P<0.01),且PC组、MD组和HD组均显著低于LD组(P<0.01),HD组均显著低于PC组和MD组(P<0.01)。结论番石榴叶总三萜可减轻脓毒症大鼠炎性反应和肾组织病理变化,还可改善肾功能,推测是通过调控PI3K/AKT通路,抑制p-PI3K、p-AKT水平、下调Nrf2 mRNA及蛋白表达实现此作用的。

Objective To investigate the therapeutic effects of total triterpenoids in guava leaves on acute kidney injury(AKI)in septic rats,and to study its regulatory mechanism on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)pathway.Methods Fifty out of 60 SD rats were randomly selected to establish a sepsis model,and the other 10 rats were enrolled as BC group.The modeled rats were randomly divided into MC group,PC group,LD group,MD group and HD group.PC group was given an intraperitoneal injection of 200,000U·kg^(-1)·d^(-1) Ulinastatin.The LD group,MD group and HD group were given an intraperitoneal injection of 60,120 and 240mg·kg^(-1)·d^(-1)total triterpenoids in guava leaves,respectively,for 1 consecutive week.Enzyme-linked immunosorbent test(ELISA)was used to detect serum creatinine(Scr)and blood urea nitrogen(BUN)levels before and after drug administration,and ELISA was used to detect the levels of serum interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α).Hematoxylin-eosin staining(HE)was used to observe the pathological changes of kidney tissue,real-time fluorescent quantitative polymerase chain reaction(RT-PCR)was used to detect mRNA expression of PI3K,AKT,and nuclear factor-2 related factors 2(Nrf2).Western blotting(WB)was used to detect PI3K,AKT,Nrf2 protein expression and phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p-AKT)levels.Results The serum levels of Scr,BUN,IL-1β,IL-18,and TNF-αlevels in BC group did not change significantly after drug administration(P>0.05),however,which in MC group were significantly increased,as compared with those before drug administration(P<0.01),moreover,which in the other 4 groups were significantly lower than those before administration(P<0.01).Before drug administration,the above indexes in MC group,PC group and 3 different drug dose groups were significantly higher than those in BC group(P<0.01).After administration,the serum levels of these indexes in MC group were significantly higher than those in BC group(P<0.05),however,which in PC group and 3 different drug dose groups were significantly lower than those in MC group(P<0.01),moreover,which in PC group,MD group and HD group were significantly lower than those in LD group(P<0.01),which in HD group were significantly lower than those in MD group(P<0.01).Conclusion Total triterpenoids in guava leaves can alleviate the inflammatory response and pathological changes of renal tissue in septic rats,and can also improve renal function,possibly by regulating the PI3K/AKT pathway,inhibiting the levels of p-PI3K and p-AKT and downregulating Nrf2 mRNA and protein expression.