摘要
目的:通过建立肝损伤与肝阴虚证大鼠模型,研究鳖血制柴胡抑制“劫肝阴”之偏性及对肝损伤肝阴虚证大鼠的保护作用。方法:将SPF级SD雄性大鼠随机分为空白组,40%、30%、20%四氯化碳(CCl_(4))组,采用腹腔注射CCl_(4)联合灌胃温热中药(附子、肉桂、干姜)的方法制备肝损伤与肝阴虚证大鼠模型。将SPF级SD大鼠随机分空白组,模型组,一贯煎组(0.7 g·kg^(-1)),生柴胡与鳖血柴胡高、中、低剂量组(1.0、2.0、4.0 g·kg^(-1))。造模第3周后给药,观测大鼠活动、体温、体质量、饮水量,取材后采用全自动生化仪分析大鼠血清中天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(AKP)、总胆红素(TBiL)、直接胆红素(DBiL)、γ-谷氨酰基转移酶(γ-GT)含量,酶联免疫吸附测定法(ELISA)检测大鼠血清中甲状腺素(T4)、醛固酮(ALD)、环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)的含量,酶标仪检测大鼠肝组织匀浆丙二醛(MDA)、超氧化物歧化酶(SOD)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)的含量,观察肝组织苏木素-伊红(HE)染色病理切片,蛋白免疫印迹法(Western blot)检测大鼠肝组织磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)蛋白表达。结果:与空白组比较,30%CCl_(4)组大鼠肛温明显升高(P<0.05),体质量显著降低(P<0.01);20%CCl_(4)组大鼠饮水量、肛温显著升高(P<0.01),体质量明显降低(P<0.05);CCl_(4)各组大鼠血清中ALT、AST、γ-GT、DBil、TBil、T3、T4、ALD、cAMP、cGMP含量均明显升高(P<0.05,P<0.01),其中T3、T4、ALD为20%CCl_(4)组升高最为显著,20%CCl_(4)组大鼠肝脏损伤较其余CCl_(4)组轻,肝小叶粘连范围不大,HE病理切片显示细胞坏死量较少,脂肪变性较轻,嗜中性粒细胞较少,有纤维化出现,但未形成假小叶。与空白组比较,一贯煎组,生柴胡和鳖血柴胡高、中剂量组大鼠体质量、肛温无显著升高;生柴胡与鳖血柴胡等剂量组之间无差异。与模型组比较,一贯煎组、生柴胡、鳖血柴胡各剂量组大鼠ALT、AST、AKP、TBil、T4、ALD、cAMP、cGMP明显降低(P<0.05,P<0.01);鳖血柴胡组大鼠SOD、MDA含量明显升高(P<0.05,P<0.01);一贯煎组、生柴胡和鳖血柴胡高中低剂量组IL-6、TNF-α水平显著下降(P<0.01),IL-10显著升高(P<0.01);生柴胡和鳖血柴胡高中低剂量组大鼠肝组织中PI3K、Akt蛋白表达量显著下调(P<0.01)。鳖血柴胡与同剂量生柴胡组对比,鳖血柴胡低剂量组对IL-6、TNF-α显著降低(P<0.01),IL-10显著升高(P<0.01);鳖血柴胡各剂量组cAMP、cGMP显著降低(P<0.01);鳖血柴胡组T4、ALD含量更接近于空白组;鳖血柴胡各剂量组PI3K蛋白表达水平明显降低(P<0.05,P<0.01)。结论:20%CCl_(4)联合温热中药模型实验后期大鼠出现易怒狂躁的现象,阴虚虚热指标显著性高,肝损伤较轻,较符合肝阴虚证的临床表现。鳖血柴胡有效治疗肝损伤肝阴虚大鼠且疗效总体上优于生柴胡,提示鳖血制柴胡能挫缓其劫肝阴之性,有效改善机体阴虚虚热症候,增强对肝脏的保护作用,其机制与抑制PI3K、Akt通路相关蛋白表达有关。
Objective:To investigate the inhibitory effect of Bupleuri Radix processed with Trionyx sinensis blood on the depletion of liver Yin and its protective effect in a rat model of liver injury with liver Yin deficiency syndrome.Methods:SPF male Sprague-Dawley(SD)rats were randomly divided into blank,40%,30%,and 20%CCl_(4) groups.Liver injury with liver Yin deficiency syndrome were induced by intraperitoneal injection of CCl_(4) combined with intragastric administration of warming Chinese medicines(Aconiti Radix Lateralis Praeparata,Cinnamomi Cortex,and Zingiberis Rhizoma).SPF SD rats were then randomly assigned to blank group,model group,Yiguanjian group(0.7 g·kg^(-1)),high-,medium-,and low-dose raw Bupleuri Radix groups,and high-,medium-,and low-dose Bupleuri Radix processed with T.sinensis blood groups(1.0,2.0,4.0 g·kg^(-1)).From the third week,treatments were administered.Rat activity,anal temperature,body weight,and water intake were monitored.Serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(AKP),total bilirubin(TBiL),direct bilirubin(DBiL),andγ-glutamyltransferase(γ-GT)were measured using an automatic biochemical analyzer.Thyroxine(T4),aldosterone(ALD),cyclic adenosine monophosphate(cAMP),and cyclic guanosine monophosphate(cGMP)levels were measured by enzyme-linked immunosorbent assay(ELISA).Liver homogenates were assayed for malondialdehyde(MDA),superoxide dismutase(SOD),interleukin-6(IL-6),interleukin-10(IL-10),and tumor necrosis factor-α(TNF-α).Liver pathology was assessed by hematoxylin-eosin(HE)staining,and phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt)protein expression in liver tissue was determined by Western blot.Results:Compared with the blank group,the 30%CCl_(4) group showed significantly increased anal temperature(P<0.05)and decreased body weight(P<0.01).The 20%CCl_(4) group exhibited significantly increased water intake and anal temperature(P<0.01)and decreased body weight(P<0.05).All CCl_(4) groups had significantly elevated serum ALT,AST,γ-GT,DBil,TBil,T3,T4,ALD,cAMP,and cGMP levels(P<0.05,P<0.01),with T3,T4,and ALD increasing most prominently in the 20%CCl_(4) group.Liver injury in the 20%CCl_(4) group was milder,with limited hepatic lobule adhesion.HE staining revealed fewer necrotic cells,milder fatty degeneration,fewer neutrophils,and fibrosis without pseudolobule formation.Compared with the blank group,the Yiguanjian group,high-and medium-dose raw Bupleuri Radix groups,and high-and medium-dose Bupleuri Radix processed with T.sinensis blood groups showed no significant increase in body weight or anal temperature,and there was no difference between groups receiving equivalent doses of Raw Bupleuri Radix and Bupleuri Radix processed with T.sinensis blood.Compared with the model group,the Yiguanjian group,raw Bupleuri Radix groups,and Bupleuri Radix processed with T.sinensis blood groups showed significant reductions in ALT,AST,AKP,TBil,T4,ALD,cAMP,and cGMP(P<0.05,P<0.01).The Bupleuri Radix processed with T.sinensis blood groups had significantly increased SOD and MDA levels(P<0.05,P<0.01).All treatment groups exhibited decreased IL-6 and TNF-α(P<0.01)and increased IL-10(P<0.01).PI3K and Akt protein expression in liver tissues was significantly downregulated in the high-,medium-,and low-dose raw Bupleuri Radix groups,and Bupleuri Radix processed with T.sinensis blood groups(P<0.01).Compared with the equivalent raw Bupleuri Radix group,the low-dose Bupleuri Radix processed with T.sinensis blood group showed lower IL-6 and TNF-α(P<0.01)and higher IL-10(P<0.01).All doses of Bupleuri Radix processed with T.sinensis blood significantly decreased cAMP and cGMP(P<0.01),and T4 and ALD levels were closer to those of the blank group.PI3K protein expression was significantly reduced in all doses of Bupleuri Radix processed with T.sinensis blood groups(P<0.05,P<0.01).Conclusion:Rats in the 20%CCl₄combined with warming Chinese medicine model exhibited irritability and mania in the late stage,with significant Yin deficiency-heat indicators and mild liver injury,reflecting clinical liver Yin deficiency syndrome.Bupleuri Radix processed with T.sinensis blood effectively protected against liver injury with liver Yin deficiency syndrome,with superior overall efficacy compared to raw Bupleuri Radix.These findings suggest that Bupleuri Radix processed with T.sinensis blood can mitigate its tendency to impair liver Yin,effectively alleviate symptoms of Yin deficiency and internal heat,and enhance its protective effect on the liver.Its mechanism is associated with inhibition of PI3K and Akt protein expression.
作者
李清宋
乔欣
候梦雨
马越兴
崔阳
叶耀辉
LI Qingsong;QIAO Xin;HOU Mengyu;MA Yuexing;CUI Yang;YE Yaohui(Jiangxi University of Chinese Medicine,Nanchang 330004,China;Nanchang Medical College,Nanchang 330052,China;Jiangxi Provincial Education Department Key Laboratory for the Application of Key Technologies in Drug Screening for Inflammatory Diseases and Phlegm Syndrome,Nanchang 330052,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第23期63-74,共12页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(H2805-82060717)
江西省卫生健康委科技创新重点项目(2025ZD001)
2022科技创新团队项目(NYTD202202)
江西省技术创新引导类计划项目(2023KZI01003)
江西省中医药标委会标准化项目(2024A001)
新疆维吾尔自治区重点研发计划项目(2022B02058)。
关键词
肝阴虚
肝损伤
柴胡
鳖血柴胡
liver Yin deficiency
liver damage
Bupleuri Radix
Bupleuri Radix processed with Trionyx sinensis blood
