麦角甾醇预处理对丙泊酚麻醉大鼠海马CA1区神经元损伤的影响

Effect of ergosterol pretreatment on neuronal damage in hippocampal CA1 area of propofol anesthetized rats

目的探讨麦角甾醇对丙泊酚麻醉大鼠海马CA1区神经元的保护作用及机制。方法将45只SD大鼠按随机数字表法分为对照组、丙泊酚组和丙泊酚+麦角甾醇组,每组15只。对照组大鼠腹腔注射100 mg/kg脂肪乳溶剂;丙泊酚组大鼠先腹腔注射50 mg/kg丙泊酚,待翻正反射恢复后再次注射50 mg/kg丙泊酚;丙泊酚+麦角甾醇组大鼠腹腔注射30 mg/kg麦角甾醇,随后给予丙泊酚注射(方法同上),均连续注射7 d。末次注射后大鼠均清醒2 h。采用透射电镜观察大鼠海马CA1区神经元的超微结构;采用HE染色、TUNEL和NeuN染色、免疫组化染色分别检测大鼠海马CA1区神经元的形态学变化、凋亡、突触后密度蛋白95(PSD95)的表达;采用Western blotting法检测大鼠海马CA1区凋亡相关蛋白和磷脂酰肌醇-3-激酶-蛋白激酶B(PI3K-Akt)信号通路蛋白的表达。结果透射电镜、HE染色结果显示丙泊酚+麦角甾醇组大鼠海马CA1区神经元损伤程度较丙泊酚组减轻。与对照组比较,丙泊酚组大鼠海马CA1区神经元凋亡率升高,活化半胱氨酸天冬氨酸蛋白酶3(Caspase 3)、Bax蛋白的表达升高,Bcl-2蛋白、PSD95的表达降低,凋亡诱导因子(AIF)、细胞色素C(Cyt-C)蛋白的表达升高,而Sirt1蛋白表达、磷酸化(p)-PI3K/PI3K和p-Akt/Akt值降低,差异均有统计学意义(P<0.05)。与丙泊酚组比较,丙泊酚+麦角甾醇组大鼠海马CA1区神经元凋亡率降低[(27.33±1.37)%vs.(17.47±0.87)%],差异有统计学意义(P<0.05)。与丙泊酚组比较,丙泊酚+麦角甾醇组大鼠海马CA1区活化Caspase 3、Bax蛋白的表达降低,Bcl-2蛋白、PSD95的表达升高,AIF、Cyt-C蛋白的表达降低,而Sirt1蛋白表达、p-PI3K/PI3K和p-Akt/Akt值升高,差异均有统计学意义(P<0.05)。结论麦角甾醇预处理可抑制丙泊酚诱导的海马CA1区神经元凋亡并减轻神经元损伤,其机制可能由PI3K-Akt信号通路介导。

Objective To investigate the protective effect of ergosterol on neurons in CA1 area of the hippocampus and its mechanism in rats anesthetized with propofol.Methods Forty-five SD rats were randomly divided into control group,propofol group and propofol+ergosterol group(n=15).Rats in the control group were injected intraperitoneally with 100 mg/kg fat emulsion solvent;rats in the propofol group were injected intraperitoneally with 50 mg/kg propofol first,and after the righting reflex was restored,they were injected with 50 mg/kg propofol;propofol+ergosterol group was intraperitoneally injected with 30 mg/kg ergosterol,followed by propofol injection,and the propofol injection method was the same as that of the propofol group.Injection was given continuously for 7 d.After the last injection,the rats in each group were awake for 2 h.The ultrastructure of neurons in hippocampal CA1 area was observed by transmission electron microscopy.HE staining,TUNEL and neuronal nuclear antigen(NeuN)staining,and Western blotting were used to detect the morphology,apoptosis,and postsynaptic density protein 95(PSD95)expression of neurons in the hippocampal CA1 area,respectively.Western blotting was used to determine the expressions of apoptosis-related proteins and phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)signaling pathway proteins in hippocampal CA1 area of rats.Results Transmission electron microscopy and HE staining showed that the damage of neurons in the hippocampal CA1 area of the propofol+ergosterol group was slighter than that of the propofol group.As compared with control group,propofol group had significantly higher neuronal apoptosis,significantly higher levels of activated Caspase 3 and Bax protein expressions,significantly decreased Bcl-2 and PSD95 expressions,significantly increased apoptosis inducing factor(AIF)and Cytochrome(Cyt)-C protein expressions,statistically lower Sirt1 protein expression,and significantly lower phosphorylated(p)-PI3K/PI3K level and p-Akt/Akt ratio in the hippocampal CA1 area(P<0.05).As compared with propofol group,propofol+ergosterol group had significantly lower neuronal apoptosis in the hippocampal CA1 area(27.33±1.37%vs.17.47±0.87%,P<0.05).As compared with propofol group,propofol+ergosterol group had significantly lower activated Caspase 3 and Bax protein expressions,significantly increased Bcl-2 and PSD95 expressions,significantly decreased AIF and Cyt-C protein expressions,statistically higher Sirt1 protein expression,and significantly higher p-PI3K/PI3K level and p-Akt/Akt ratio in the hippocampal CA1 area(P<0.05).Conclusion Ergosterol pretreatment can inhibit propofol-induced neuron apoptosis and alleviate the neuronal damage in the hippocampal CA1 area,whose mechanism may be mediated by PI3K-Akt signaling pathway.