健脾疏肝丸对非酒精性脂肪性肝病大鼠肝X受体α-固醇调节元件结合蛋白-1-脂肪酸合成酶信号转导通路的影响

Effects of Jianpi Shugan Pill on liver X receptorα-sterolregulatory element-binding protein-1-fatty acid synthase signaling transduction pathway in rats with non-alcoholic fatty liver disease

目的探讨健脾疏肝丸对非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)大鼠肝X受体(liver X receptorα,LXRα)-固醇调节元件结合蛋白-1(sterolregulatory element-binding protein-1,SREBP-1)-脂肪酸合成酶(fatty acid synthase,FAS)信号转导通路的影响。方法将32只无特定病原体(specific pathogen free,SPF)SD雄性大鼠按照随机数字表法分为正常组、模型组、健脾疏肝丸组和易善复组,每组8只。正常组进食普通饲料,其他组进食高脂饲料,健脾疏肝丸组给予4.86 g/(kg·d)灌胃,易善复组给予0.123 g/(kg·d)灌胃,正常组和模型组给予等量蒸馏水灌胃。灌胃与造模同时进行,共8周。检测大鼠血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、高密度脂蛋白(high-density lipoprotein cholesterol,HDL-C)、低密度脂蛋白(low-density lipoprotein cholesterol,LDL-C)、甘油三酯(triglycerides,TG)、总胆固醇(total cholesterol,TC)、肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)及白细胞介素-6(interleukin-6,IL-6)水平。对大鼠肝组织切片进行HE染色和油红O染色,于光学显微镜下观察。采用免疫组织化学法、蛋白质免疫印迹法及实时荧光定量聚合酶链式反应(real time polymerase chain reaction,RT-PCR)检测大鼠LXRα、SREBP-1及FAS的表达水平。结果 (1)正常组、模型组、健脾疏肝丸组和易善复组大鼠ALT[(3.40±0.81)U/L vs(9.98±2.27)U/L vs(7.80±1.52)U/L vs(6.43±1.89)U/L]、AST [(10.61±1.17)U/L vs(23.63±4.82)U/L vs(18.04±2.98)U/L vs(16.42±3.30)U/L]、TNF-α[(2.40±0.96)×10-3μg/L vs(6.64±0.92)×10-3μg/L vs(4.87±1.35)×10-3μg/L vs(4.45±1.39)×10-3μg/L]、IL-6 [(0.95±0.81)pg/ml vs(7.88±3.08)pg/ml vs(3.17±1.26)pg/ml vs(1.64±0.55)pg/ml]、TG [(0.33±0.13)mmol/L vs(0.90±0.24)mmol/L vs(0.62±0.37)mmol/L vs 0.62(0.46,0.66)mmol/L]、TC [(2.10±0.42)mmol/L vs 5.34(5.17,6.12)mmol/Lvs(3.68±0.63)mmol/Lvs(3.41±0.81)mmol/L]、HDL-C [(1.07±0.17)mmol/Lvs(0.62±0.14)mmol/Lvs(0.78±0.13)mmol/Lvs(0.79±0.12)mmol/L]及LDL-C[0.38(0.26,0.41)mmol/L vs(0.69±0.11)mmol/L vs(0.41±0.13)mmol/L vs(0.43±0.10)mmol/L]水平差异均有统计学意义(P <0.05)。与正常组相比,模型组AST、ALT、LDL-C、TG、TC、IL-6及TNF-α显著升高,HDL-C显著降低(P <0.05);与模型组相比,健脾疏肝丸组和易善复组AST、ALT、LDL-C、TG、TC、IL-6、TNF-α显著降低,HDL-C显著升高(P<0.05);与健脾疏肝丸组相比,易善复组大鼠血清TC显著降低(P <0.05),其他各指标差异无统计学意义(P> 0.05)。(2)肝组织HE染色和油红O染色示:与正常组相比,模型组大鼠肝脏脂肪变严重;与模型组相比,健脾疏肝丸组大鼠肝脏脂肪变减轻。(3)免疫组织化学结果表明,正常组、模型组、健脾疏肝丸组和易善复组大鼠LXRα(345872±52737 vs 544998±55506 vs 436319±65076 vs 448588±104641)、SREBP-1(259408±71143 vs 538701±62336vs 399705±102395 vs 394167±158047)和FAS(201683±48205 vs 466884±74934 vs 425589±63672 vs417852±84373)相对表达水平差异有统计学意义(P <0.05)。与正常组相比,模型组各蛋白相对表达水平均显著升高(P <0.05);与模型组相比,健脾疏肝丸组与易善复组各蛋白相对表达水平显著下降(P <0.05);健脾疏肝丸组与易善复组各蛋白相对表达水平差异无统计学意义(P> 0.05)。(4)Western blot结果表明,正常组、模型组、健脾疏肝丸组和易善复组大鼠LXRα[0.80±0.29 vs 1.57(1.30,1.67) vs 1.09±0.30 vs 1.10±0.36]、SREBP-1(0.42±0.12 vs 1.15±0.45 vs 0.86±0.20 vs 0.84±0.20)和FAS(0.43±0.12 vs 1.10±0.40 vs 0.81±0.26 vs 0.80±0.28)相对表达水平差异有统计学意义(P <0.05)。与正常组对比,模型组小鼠各蛋白相对表达水平显著升高(P <0.05);与模型组对比,健脾疏肝丸组及易善复组小鼠LXRα蛋白相对表达水平显著降低(P <0.05),健脾疏肝丸组与易善复组相比,各蛋白相对表达水平差异无统计学意义(P> 0.05)。(5)正常组、模型组、健脾疏肝丸组和易善复组大鼠肝组织LXRα[1.13±0.38 vs 4.14(4.01,4.35) vs 2.65±1.85 vs 1.35(0.54,4.23)]、SREBP-1、FAS[1.37±0.49 vs 4.35±1.97 vs 1.98(1.88,3.22)m RNA相对表达量差异有统计学意义(P <0.05)。与正常组相比,模型组LXRα[1.46±0.51 vs 6.13±1.17 vs 3.82±2.06 vs 1.56(1.19,4.74)]、SREBP-1、FAS vs1.83(1.64,4.29)] m RNA相对表达量显著升高(P <0.05);与模型组对比,健脾疏肝丸组及易善复组LXRα、SREBP-1、FAS m RNA表达均显著降低(P <0.05),健脾疏肝丸组与易善复组相比,LXRα、SREBP-1、FAS m RNA表达差异无统计学意义(P> 0.05)。结论健脾疏肝丸对大鼠NAFLD的改善作用可能与其抑制LXRα-SREBP-1-FAS信号转导通路有关。

Objective To investigate the effects of Jianpi Shugan Pill on liver X receptorα(LXRα)-sterolregulatory element-binding protein-1(SREBP-1)-fatty acid synthase(FAS)signaling pathway in rats with non-alcoholic fatty liver disease.Methods Total of 32 specific pathogen free(SPF)SD male rats were randomly divided into normal group,model group,Jianpi Shugan Pill group and Essentiale group according to random digital tables method,8 rats in each group.Rats in normal group were fed with normal diet,and rats in other groups were fed with high fat diet.Rats in Jianpi Shugan Pill group were given Jianpi Shugan Pill 4.86 g/(kg·d)and rats in Essentiale group were given Essentiale 0.123 g/(kg·d)by gavage.Rats in normal group and model group were giver the same amount of distilled water by gavage.Gavage and molding were carried out simultaneously for 8 weeks.Rats were sacrificed at the end of the 8 th week,and serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),triglycerides(TG),total cholesterol(TC),tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6)were detected.Liver tissue sections of rats were stained by HE and oil red O,and were observed by optical microscope.Immunohistochemical method,Western blot and real time polymerase chain reaction(RT-PCR)were used to detect the expression levels of LXRα,SREBP-1 and FAS.Results(1)ALT[(3.40±0.81)U/L vs(9.98±2.27)U/L vs(7.80±1.52)U/L vs(6.43±1.89)U/L],AST[(10.61±1.17)U/L vs(23.63±4.82)U/L vs(18.04±2.98)U/L vs(16.42±3.30)U/L],TNF-α[(2.40±0.96)×10-3μg/L vs(6.64±0.92)×10-3μg/L vs(4.87±1.35)×10-3μg/L vs(4.45±1.39)×10-3μg/L],IL-6[(0.95±0.81)pg/ml vs(7.88±3.08)pg/ml vs(3.17±1.26)pg/ml vs(1.64±0.55)pg/ml],TG[(0.33±0.13)mmol/L vs(0.90±0.24)mmol/L vs(0.62±0.37)mmol/L vs 0.62(0.46,0.66)mmol/L],TC[(2.10±0.42)mmol/L vs 5.34(5.17,6.12)mmol/L vs(3.68±0.63)mmol/L vs(3.41±0.81)mmol/L],HDL-C[(1.07±0.17)mmol/L vs(0.62±0.14)mmol/L vs(0.78±0.13)mmol/L vs(0.79±0.12)mmol/L]and LDL-C[0.38(0.26,0.41)mmol/L vs(0.69±0.11)mmol/L vs(0.41±0.13)mmol/L vs(0.43±0.10)mmol/L]of rats in normal group,model group,Jianpi Shugan Pill group and Essentiale group were statistically significant(P<0.05).Compared with normal group,AST,ALT,LDL-C,TG,TC,IL-6 and TNF-αof rats in model group increased significantly and HDL-C decreased significantly(P<0.05).Compared with model group,AST,ALT,LDL-C,TG,TC,IL-6 and TNF-αof rats in Jianpi Shugan Pill group and Essentiale group decreased significantly and HDL-C increased significantly(P<0.05).Compared with Jianpi Shugan Pill group,TC of rats in Essentiale group decreased significantly,and there were no significant differences in other indexes(P>0.05).(2)HE and oil red O staining showed that fatty lesions in liver were severe in model group than the normal group,which were alleviated in Jianpi Shugan pill group.(3)Immunohistochemical results showed that relative expression level of LXRα(345872±52737 vs 544998±55506 vs 436319±65076 vs 448588±104641),SREBP-1(259408±71143 vs 538701±62336 vs 399705±102395 vs 394167±158047)and FAS(201683±48205 vs 466884±74934 vs 425589±63672 vs 417852±84373)of rats in normal group,model group,Jianpi Shugan Pill group and Essentiale group were statistically significant(P<0.05).Compared with model group,the above relative expression levels of rats in model group increased significantly(P<0.05).Compared with model group,the above relative expression levels of rats in Jianpi Shugan Pill group and Essentiale group decreased significantly(P<0.05).There were no significant differences in the above relative expression levels of rats in Jianpi Shugan Pill group and Essentiale group(P>0.05).(4)Western blot results showed that LXRα[0.80±0.29 vs 1.57(1.30,1.67)vs 1.09±0.30 vs 1.10±0.36],SREBP-1(0.42±0.12 vs 1.15±0.45 vs 0.86±0.20 vs 0.84±0.20)and FAS(0.43±0.12 vs 1.10±0.40 vs 0.81±0.26 vs 0.80±0.28)of rats in in normal group,model group,Jianpi Shugan Pill group and Essentiale group were statistically significant(P<0.05).Compared with model group,the above relative expression levels of rats in model group increased significantly(P<0.05).Compared with model group,the LXRαrelative expression level of rats in Jianpi Shugan Pill group and Essentiale group decreased significantly(P<0.05).There were no significant differences in the above relative expression levels of rats between Jianpi Shugan Pill group and Essentiale group(P>0.05).(5)Relative expression levels of LXRα[1.13±0.38 vs 4.14(4.01,4.35)vs 2.65±1.85 vs 1.35(0.54,4.23)],SREBP-1[1.46±0.51 vs 6.13±1.17 vs 3.82±2.06 vs 1.56(1.19,4.74)]and FAS[1.37±0.49 vs 4.35±1.97 vs 1.98(1.88,3.22)vs 1.83(1.64,4.29)]m RNA of rats in normal group,model group,Jianpi Shugan Pill group and Essentiale group were statistically significant(P<0.05).Compared with model group,the relative expression levels of LXRα,SREBP-1 and FAS m RNA of rats in Jianpi Shugan Pill group and Essentiale group decreased significantly(P<0.05).There were no significant differences in the above indexes of rats in Jianpi Shugan Pill group and Essentiale group(P>0.05).Conclusions The therapeutic effect of Jianpi Shugan Pill on NAFLD rats may be related to its obstruction of LXRα-SREBP-1-FAS signaling transduction pathway.