银蓝调脂胶囊通过调节自噬治疗非酒精性脂肪肝的机制研究

Study on Songyu Anshen Prescription Improving Learning and Memory Ability of Chronic SleepDeprived Rats Through NLRP3 Inflammasome Pathway in Hippocampus

目的探讨银蓝调脂胶囊调控自噬相关信号通路对非酒精性脂肪肝(Non-alcoholic fatty liver disease,NAFLD)治疗的机制。方法选取60只小鼠,随机抽取10只作为正常对照组,余下高脂饲料喂养12周建立NAFLD模型。模型复制成功后,将造模组分成模型组、非诺贝特组及银蓝调脂胶囊高、中、低剂量组,每组10只。各给药组按剂量分别灌胃相应药物30 d进行干预。给药结束后检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、谷丙转氨酶(ALT)、谷草转氨酶(AST)含量;取整肝计算肝脏系数;苏木精-伊红(HE)和油红O染色观察各组小鼠肝脂肪变性程度及组织学特征;Elisa试剂盒检测血清炎症因子白细胞介素6(IL-6)、肿瘤坏死因子(TNF)-α,氧化应激相关丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;采用试剂盒检测肝脏载脂蛋白(Apo)A1、ApoB、ApoE的水平;Western Blot法检测脂质生成关键蛋白SREBF-1、FAS及ACC1的蛋白表达,检测自噬通路Beclin1、ATG3、ATG5、ATG7、P62的蛋白表达水平。结果与正常对照组比较,模型组小鼠肝脏存在明显脂肪样变性,体质量、肝脏系数及血清TC、TG、LDL-C、ALT、AST、MDA含量均明显升高(P<0.01),血清中促炎因子IL-6、TNF-α含量明显增加(P<0.01),HDL-C、SOD、GSH-Px水平明显降低(P<0.01);肝脏组织中ApoA1含量明显降低(P<0.01),ApoB、ApoE含量明显升高(P<0.01);肝组织中脂肪合成蛋白SREBF-1、FAS及ACC1表达升高(P<0.01),小鼠肝脏中P62的表达升高而Beclin1、ATG3、ATG5蛋白表达水平降低(P<0.05,P<0.01)。经过治疗后,非诺贝特组及银蓝调脂胶囊各剂量组小鼠的脂肪变性得到改善,肝脏系数、血清中TC、TG、LDL-C、ALT、AST、MDA、IL-6、TNF-α含量均明显降低(P<0.01),HDL-C、SOD、GSH-Px水平明显升高(P<0.01),ApoB、ApoE含量明显降低(P<0.01),肝脏脂肪合成蛋白ACC1表达明显降低(P<0.01),肝脏P62蛋白表达降低(P<0.05,P<0.01);非诺贝特组及银蓝调脂胶囊高、中剂量组肝脏组织中ApoA1含量明显升高(P<0.05),SREBF-1、FAS蛋白表达降低(P<0.01),ATG5、Beclin1蛋白表达升高(P<0.05,P<0.01);非诺贝特组及银蓝调脂胶囊高剂量组ATG3、ATG7蛋白表达升高(P<0.05,P<0.01)。结论银蓝调脂胶囊通过调节自噬相关通路改善脂肪变性从而治疗非酒精性脂肪肝。

Objective To investigate the effects and mechanisms of Songyu Anshen Prescription(Nardostachyos Radix et Rhizoma,Curcumae Radix,Salviae Miltiorrhizae Radix et Rhizoma,Ziziphi Spinosae Semen,etc.)to improve the learning and memory ability of chronic sleep-deprived rats through the hippocampal NOD-like receptor thermoprotein structural domain-related protein 3(NLRP3)inflammasome pathway.Methods SD rats were randomly divided into normal(8 mice),large platform(8 mice)and sleep deprivation(32 mice)groups,and a modified multi-platform water environment method was used to replicate the chronic sleep deprivation rat model for21 consecutive days.After the replication was successful,the sleep deprivation rats were randomly divided into the model group and the high-,medium-and low-dose groups of Songyu Anshen Prescription(34,17,and 8.5 g·kg(-1)),with 8 rats in each group;the rats in all groups were administered by gavage once a day for 7 days.Changes in the general state and body mass of rats were observed and recorded during the modeling and drug administration periods;the learning and memory ability of rats was measured on the day 21 of modeling and on the day 7 of drug administration by using the Morris water maze method(local navigation and spatial search test);after the final water maze test,interleukin 1β(IL-1β)and IL-18 were detected in hippocampal tissue by using the ELISA method;Western Blot method was used to detect the protein expression levels of NLRP3,apoptosis-associated speck-like protein(ASC)and cysteine aspartate protease 1(Caspase-1)in hippocampal tissue.Results(1)Compared with the normal group and the large platform group,there was no significant change in all the detected indexes(P>0.05).(2)Compared with the large platform group,the body mass of rats in the sleep deprivation group increased slowly during the modeling period(P<0.01);after 21 days of sleep deprivation,rats in the sleep deprivation group had a significantly longer evasion latency(P<0.01),a significant increase in the total distance to reach the platform(P<0.01),and a significant decrease in the number of times they traversed the platform and the time they stayed in the target quadrant(P<0.05,P<0.01);the contents of IL-1βand IL-18 in the hippocampal tissue of rats in the model group were significantly increased(P<0.05),and the protein expressions of NLRP3,ASC,and Caspase-1 were all significantly up-regulated(P<0.05).(3)Compared with the model group,the increase in body mass of rats in both the high-and medium-dose groups of Songyu Anshen Prescription was significantly increased(P<0.01);the evasion latency was significantly shortened(P<0.05),the total distance to the platform was significantly shortened(P<0.05),the number of times of traversing the platform and the time of staying in the target quadrant were significantly increased(P<0.05,P<0.01);and the hippocampal tissue had significantly higher IL-1β,IL-18 contents were significantly decreased(P<0.05,P<0.01),and protein expressions of NLRP3,ASC,and Caspase-1 were significantly down-regulated(P<0.05,P<0.01).Conclusion Songyu Anshen Prescription may regulate the secretion of pro-inflammatory cytokines IL-1βand IL-18 by inhibiting the NLRP3 inflammasome pathway,therefore improve the learning and memory ability of sleep-deprived rats.