银杏内酯K通过AMPK介导的能量代谢和自噬缓解创伤性脑损伤

Ginkgolide K Alleviates Traumatic Brain Injury through AMPK-mediated Energy Metabolism and Autophagy

目的:探讨银杏内酯K(GK)对创伤性脑损伤(TBI)大鼠的治疗作用,并分析其对AMP活化蛋白激酶(AMPK)活性的影响。方法:将大鼠分为Sham组、TBI组、L-GK组、M-GK组、H-GK组、H-GK+CC组(n=11)。Sham组大鼠为假手术大鼠,其他组大鼠为颅脑损伤仪制作的TBI模型大鼠。Sham组和TBI组大鼠灌胃0.5%羧甲基纤维素钠。L-GK组、M-GK组、H-GK组大鼠分别灌胃剂量为2、4和8 mg/kg/d的银杏内酯K。H-GK+CC组大鼠灌胃剂量8 mg/kg/d的银杏内酯K,并腹腔注射剂量为20mg/kg/d的AMPK抑制剂Compound C(CC)。各组大鼠均给药14 d。通过改良神经功能严重程度评分(m NSS)评价大鼠神经功能,采用Morris水迷宫实验评价大鼠认知功能,记录逃避潜伏期(EL)和1 min内穿越平台次数(NPC)。分别对大鼠脑组织切片进行HE、Nissl和TUNEL染色以及Bax和Bcl-2免疫组化染色。按照试剂盒说明检测大鼠脑组织SOD、CAT、GSH-Px、MDA、IL-1β、IL-6和TNF-α水平,Na+-K+-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性以及ATP含量。采用比色法检测血清NO和T-NOS水平。通过Western blot检测大鼠脑组织p-AMPKα(Thr172)、p62和Beclin-1蛋白表达水平。结果:与Sham组比较,TBI组大鼠的m NSS评分和EL升高(P<0.05),NPC降低(P<0.05);HE染色评分升高(P<0.05);TUNEL阳性率和Bax染色评分升高(P<0.05),Bcl-2染色评分降低(P<0.05);MDA、IL-1β、IL-6、TNF-α、NO和T-NOS水平升高(P<0.05),SOD、CAT和GSH-Px水平降低(P<0.05);Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性以及ATP含量降低(P<0.05),p-AMPKα(Thr172)和Beclin-1蛋白相对表达水平降低,p62蛋白相对表达水平升高(P<0.05)。与TBI组比较,L-GK组、M-GK组和H-GK组大鼠的m NSS评分和EL降低(P<0.05),NPC升高(P<0.05);HE染色评分降低(P<0.05);TUNEL阳性率和Bax染色评分降低(P<0.05),Bcl-2染色评分升高(P<0.05);MDA、IL-1β、IL-6、TNF-α、NO和T-NOS水平降低(P<0.05),SOD、CAT和GSH-Px水平升高(P<0.05);Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性以及ATP含量升高(P<0.05),p-AMPKα(Thr172)和Beclin-1蛋白相对表达水平升高(P<0.05),p62蛋白相对表达水平降低(P<0.05)。与H-GK组比较,H-GK+CC组大鼠的m NSS评分和EL升高(P<0.05),NPC降低(P<0.05);HE染色评分升高(P<0.05);TUNEL阳性率和Bax染色评分升高(P<0.05),Bcl-2染色评分降低(P<0.05);MDA、IL-1β、IL-6、TNF-α、NO和T-NOS水平升高(P<0.05),SOD、CAT和GSH-Px水平降低(P<0.05);Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性以及ATP含量降低(P<0.05),p-AMPKα(Thr172)和Beclin-1蛋白相对表达水平降低(P<0.05),p62蛋白相对表达水平升高(P<0.05)。结论:银杏内酯K通过改善AMPK介导的能量代谢和自噬缓解TBI大鼠的脑损伤。

Objective:To explore the possible role of ginkgolide K(GK)in the treatment of traumatic brain injury(TBI)and analyze its effect on AMP-activated protein kinase(AMPK)activity.Methods:Rats were divided into Sham group,TBI group,L-GK group,M-GK group,H-GK group,and H-GK+CC group(n=11).Rats in Sham group was sham-operated rats,and rats in other groups were TBI model rats which made by craniocerebral injury instrument.Rats in Sham group and TBI group were administered 0.5%CMC-Na by intragastric administration.Rats in L-GK group,M-GK group,and H-GK group were administered 2,4,and 8 mg/kg/d ginkgolide K respectively.Rats in H-GK+CC group were given with 8 mg/kg/d Ginkgolide K and intraperitoneally injected with 20mg/kg/d of AMPK inhibitor Compound C(CC).Rats were all dosed for 14 days.Neurological function was assessed by the modified Neurological Severity Score(mNSS).The Morris water maze test was used to evaluate cognitive function,and the escape latency(EL)and the number of platform crossings within 1 min(NPC)were recorded.Brain tissue sections were subjected to HE,Nissl and TUNEL staining as well as Bax and Bcl-2 immunohistochemical staining.The levels of SOD,CAT,GSH-Px,MDA,IL-1β,IL-6 and TNF-α,the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase and the content of ATP in brain tissue were detected according to the kit instructions.The serum levels of NO and T-NOS were detected by colorimetry.The protein expression levels of p-AMPKα(Thr172),p62 and Beclin-1 in brain tissue were detected by Western blot.Results:Compared with Sham group,the m NSS score and EL of TBI group increased(P<0.05),the NPC decreased(P<0.05),the HE staining score increased(P<0.05),the TUNEL positivity rate and Bax staining score increased(P<0.05),the Bcl-2 staining score decreased(P<0.05),the levels of MDA,IL-1β,IL-6,TNF-α,NO and T-NOS increased(P<0.05),the levels of SOD,CAT and GSH-Px decreased(P<0.05),the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase and ATP content decreased(P<0.05),the relative expression levels of p-AMPKα(Thr172)and Beclin-1 protein decreased(P<0.05),and the relative expression levels of p62 protein increased(P<0.05).Compared with TBI group,the m NSS score and EL of L-GK,M-GK and H-GK groups decreased(P<0.05),the NPC increased(P<0.05),the HE staining score decreased(P<0.05),the TUNEL positive rate and Bax staining score decreased(P<0.05),the Bcl-2 staining score increased(P<0.05),the levels of MDA,IL-1β,IL-6,TNF-α,NO and T-NOS decreased(P<0.05),the levels of SOD,CAT and GSH-Px increase(P<0.05),the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase and ATP content increased(P<0.05),the relative expression level of p-AMPKα(Thr172)and Beclin-1 protein increased(P<0.05),and the relative expression level of p62 protein decreased(P<0.05).Compared with H-GK group,the m NSS score and EL of H-GK+CC group increased(P<0.05),the NPC decreased(P<0.05),the HE staining score increased(P<0.05),the TUNEL positivity rate and Bax staining score increased(P<0.05),the Bcl-2 staining score decreased(P<0.05),the levels of SOD,CAT and GSH-Px decreased(P<0.05),the levels of MDA,IL-1β,IL-6,TNF-α,NO and T-NOS increased(P<0.05),the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase and ATP content decreased(P<0.05),the relative expression levels of p-AMPKα(Thr172)and Beclin-1 protein decreased(P<0.05),and the relative expression levels of p62 protein increased(P<0.05).Conclusion:Ginkgolide K could alleviate brain damage in TBI rats by improving AMPK-mediated energy metabolism and autophagy.