丹参提取物对脑外伤模型大鼠脑神经元SIRT1-FoxO1-自噬通路的影响

Effect of Salvia Miltiorrhiza Extract on SIRT1-FoxO1-Autophagy Pathway in Neurons of Rats with Cerebral Trauma

目的研究丹参提取物(SME)对脑外伤大鼠神经元沉默信息调节因子2相关酶1(SIRT1)-叉头框蛋白O1(FoxO1)-自噬通路的影响。方法采用改良Feeney法构建大鼠脑外伤模型,将78只SD大鼠按照随机数字表法分成假手术组、模型组、SME低、中、高剂量组和抑制剂组,每组13只。假手术组和模型组大鼠腹腔注射生理盐水2mL,SME低、中、高剂量组大鼠分别按0.75、1.50、3.00mg/kg给予SME,抑制剂组予SME中剂量组剂量给药同时按照5mg/kg注射SIRT1抑制剂(EX-527)。通过尼氏染色观察大鼠右侧脑组织神经元损伤情况;免疫组织化学染色、逆转录聚合酶链式反应(RT-PCR)、蛋白免疫印迹法(Western blot)分别测定SIRT1和FoxO1的细胞阳性率、转录(mRNA)水平和蛋白表达水平。结果与假手术组比较,模型组SIRT1和FoxO1的阳性细胞比例、mRNA和蛋白表达量均显著升高[(12.29±2.42)%比(4.36±1.21)%,(13.15±1.67)%比(3.31±1.50)%,(0.87±0.06)比(0.62±0.05),(0.89±0.03)比(0.69±0.04),(0.72±0.04)比(0.43±0.04),(0.76±0.08)比(0.31±0.09),P<0.05];与模型组比较,抑制剂组SIRT1阳性细胞比例、mRNA和蛋白表达量均显著降低[(6.88±2.12)%比(12.29±2.42)%,(0.71±0.09)比(0.87±0.06),(0.54±0.08)比(0.72±0.04),P<0.05],SME低、中剂量两组SIRT1蛋白水平升高[(0.98±0.05)、(1.12±0.08)比(0.72±0.04),P<0.05]及SME高剂量组SIRT1、FoxO1阳性细胞比例和mRNA和蛋白表达量均显著升高[(19.04±1.81)%比(12.29±2.42)%,(18.09±1.34)%比(13.15±1.67)%,(1.21±0.10)比(0.87±0.06),(1.19±0.03)比(0.89±0.03),(1.15±0.13)比(0.72±0.04),(0.95±0.09)比(0.76±0.08),P<0.05];与抑制剂组比较,SME中、高剂量两组FoxO1阳性细胞比例升高[(14.63±2.22)%、(18.09±1.34)%比(9.85±1.31)%,P<0.05],SME低、中、高剂量三组SIRT1阳性细胞比例、SIRT1、FoxO1的mRNA和SIRT1蛋白水平都显著升高[(12.52±2.01)%、(14.82±2.11)%、(19.04±1.81)%比(6.88±2.12)%,(0.86±0.08)、(0.92±0.05)、(1.21±0.10)比(0.71±0.09),(0.89±0.06)、(0.98±0.07)、(1.19±0.03)比(0.82±0.05),(0.98±0.05)、(1.12±0.08)、(1.15±0.13)比(0.54±0.08),P<0.05];与SME低剂量组比较,SME中剂量组FoxO1 mRNA和SIRT1蛋白表达量[(0.98±0.07)比(0.89±0.06),(1.12±0.08)比(0.98±0.05),P<0.05]及SME高剂量组SIRT1阳性细胞比例、SIRT1和FoxO1的mRNA和蛋白表达量均显著升高[(19.04±1.81)%比(12.52±2.01)%,(1.21±0.10)比(0.86±0.08),(1.19±0.03)比(0.89±0.06),(1.15±0.13)比(0.98±0.05),(0.95±0.09)比(0.79±0.07),P<0.05];与SME中剂量组比较,SME高剂量组SIRT1和FoxO1 mRNA表达显著升高[(1.21±0.10)比(0.92±0.05),(1.19±0.03)比(0.98±0.07),P<0.05]。结论SME能够明显促进脑外伤模型大鼠脑神经元SIRT1表达,增强SIRT1的去乙酰化活性,正向调节SIRT1-FoxO1-自噬通路发挥神经保护作用。

Objective To investigate the effect of Salvia miltiorrhiza extract(SME)on silence information regulatorprotein 1(SIRT1)-forkhead box O1(FoxO1)-autophagy pathway in neurons of rats with cerebral trauma.Methods The modified Feeney method was used to construct a rat model with cerebral trauma.Seventy-eight SD rats were randomly divided into sham operation group,model group,low,medium and high dose of SME group,and inhibitor group,with 13 animals in each group.Rats in the sham group and the model group were injected with 2mL of normal saline intraperitoneally;rats in low,medium and high dose of SME groups were given 0.75,1.50 and 3.00mg/kg SME,respectively;rats in the inhibitor group were given both medium dose of SME and 5mg/kg SIRT1 inhibitor(EX-527).The neuronal damage in the right brain tissue of rats was observed by Nissl staining.Immunohistochemical staining,reverse transcription polymerase chain reaction(RT-PCR)and Western blot were used to measure the cell positive rate,transcription(mRNA)level and protein expression level of SIRT1 and FoxO1,respectively.Results Compared to the sham group,the positive cell rate,mRNA and protein expression of SIRT1 and FoxO1 in the model group increased significantly,i.e.,positive cell rate of SIRT1:[(12.29±2.42)%vs(4.36±1.21)%],positive cell rate of FoxO1:[(13.15±1.67)%vs(3.31±1.50)%],SIRT1 mRNA:[(0.87±0.06)vs(0.62±0.05)],FoxO1 mRNA:[(0.89±0.03)vs(0.69±0.04)],SIRT1 protein:[(0.72±0.04)vs(0.43±0.04)],FoxO1 protein:[(0.76±0.08)vs(0.31±0.09)].Compared to the model group,the positive cell rate,mRNA and protein expression of SIRT1 in the inhibitor group decreased significantly,i.e.,[(6.88±2.12)%vs(12.29±2.42)%],[(0.71±0.09)vs(0.87±0.06)],[(0.54±0.08)vs(0.72±0.04)];SIRT1 protein level in low and medium dose group increased,i.e.,[(0.98±0.05)and(1.12±0.08)vs(0.72±0.04)];positive cell rate,mRNA and protein expression of SIRT1 and FoxO1 in high dose group increased,i.e.,[(19.04±1.81)%vs(12.29±2.42)%],[(18.09±1.34)%vs(13.15±1.67)%],[(1.21±0.10)vs(0.87±0.06)],[(1.19±0.03)vs(0.89±0.03)],[(1.15±0.13)vs(0.72±0.04)],[(0.95±0.09)vs(0.76±0.08)].Compared to the inhibitor group,positive cell rate of FoxO1 in medium and high dose group increased,i.e.,[(14.63±2.22)%and(18.09±1.34)%vs(9.85±1.31)];positive cell rate of SIRT1,SIRT1 and FoxO1 mRNA,and SIRT1 protein in low,medium and high dose group increased significantly,i.e.,[(12.52±2.01)%,(14.82±2.11)%and(19.04±1.81)%vs(6.88±2.12)%],[(0.86±0.08),(0.92±0.05)and(1.21±0.10)vs(0.71±0.09)],[(0.89±0.06),(0.98±0.07)and(1.19±0.03)vs(0.82±0.05)],[(0.98±0.05),(1.12±0.08)and(1.15±0.13)vs(0.54±0.08)].Compared to low dose group,FoxO1 mRNA and SIRT1 protein in medium dose group increased,i.e.,[(0.98±0.07)vs(0.89±0.06)],[(1.12±0.08)vs(0.98±0.05)];positive cell rate of SIRT1,both mRNA and protein levels of SIRT1 and FoxO1 in high dose group increased,i.e.,[(19.04±1.81)%vs(12.52±2.01)%],[(1.21±0.10)vs(0.86±0.08)],[(1.19±0.03)vs(0.89±0.06)],[(1.15±0.13)vs(0.98±0.05)],[(0.95±0.09)vs(0.79±0.07)].Compared to medium dose group,SIRT1 and FoxO1 mRNA in high dose group increased,i.e.,[(1.21±0.10)vs(0.92±0.05)],[(1.19±0.03)vs(0.98±0.07)].All P<0.05,respectively.Conclusion SME can obviously promote the expression of SIRT1,enhance the deacetylation activity of SIRT1,and positively regulate SIRT1-FoxO1 autophagy pathway to play a neuroprotective role in rats with cerebral trauma.