摘要
目的观察正肝方对二乙基亚硝胺(Diethylnitrosamine,DEN)诱导的肝癌前病变大鼠的生化指标和PTEN/PI3K/AKT信号通路的影响,探讨其对肝癌前病变大鼠的作用机制。方法将80只大鼠按照随机数字表法分为5组:空白对照组、DEN模型组、正肝方低、中、高剂量组(2 g/kg、4 g/kg、8 g/kg),每组各16只。为建立肝癌前病变大鼠模型,除空白组外,其余各组大鼠连续16周每周1次腹腔注射DEN(50 mg/kg)。同时,从造模第1周开始,空白对照组及模型组灌喂纯水10 ml/kg,正肝方低、中、高剂量组分别灌喂相应剂量的正肝方药液,1次/d,连续灌胃至第16周。禁食不禁水16 h后测量大鼠体质量,麻醉,心脏取血,称量肝脏、胸腺重量。全自动生化法检测肝功能指标,包括丙氨酸氨基转移酶(Alanine aminotransferase,ALT)、碱性磷酸酶(Alkaline phosphatase,ALP)、白蛋白(Albumin,ALB)、γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,γ-GT);HE染色观察肝组织病理改变,免疫组化和蛋白质免疫印迹法(Western blot)检测肝组织中甲胎蛋白(Alpha fetoprotein,AFP)的阳性表达水平,蛋白质免疫印迹法检测肝组织中PTEN/PI3K/AKT信号通路相关蛋白表达水平。结果与正常组比较,DEN模型组大鼠体质量明显降低,肝脏指数增加,胸腺指数下降,肝功能指标除ALB外均明显升高,HE染色可见模型组肝细胞出现不同程度的变性、坏死、排列紊乱、细胞多形性等癌前病变表现,肿瘤组织中AFP、p-PI3K、p-AKT蛋白表达水平明显升高,PTEN蛋白表达水平降低,差异有统计学意义(P<0.01);与DEN模型组比较,正肝方中、高剂量组肝脏指数均有降低,高剂量组较为明显,差异有统计学意义(P<0.05),各组胸腺指数表现出不同程度升高,差异有统计学意义(P<0.05,P<0.001),血清ALT、ALP、GGT水平降低,ALB升高,肿瘤变性坏死区减少,AFP、p-PI3K、p-AKT蛋白表达水平明显降低,差异有统计学意义(P<0.01,P<0.05),PTEN蛋白表达水平提高,差异有统计学意义(P<0.01)。结论正肝方可改善肝功能指标和肝癌前病变状况,提高机体免疫力和PTEN抑癌蛋白的表达水平,降低组织中AFP、p-PI3K、p-AKT等癌症蛋白的表达,其机制可能与抑制PTEN/PI3K/AKT通路的激活,从而抑制癌前病变的发生发展相关。
Objective To observe the effects of Zhenggan Prescription on the biochemical indexes and PTEN/PI3K/AKT signaling pathway in the rat model of diethylnitrosamine(DEN)-induced precancerous lesion of the liver and to explore the mechanism of Zhenggan Prescription in treating precancerous lesion of the liver.Methods According to the random number table method,80 rats were randomized into 5 groups(n=16):blank control group,DEN model group,and low-,medium-,and high-dose(2,4,and 8 g/kg,respectively)Zhenggan Prescription groups.The rats in other groups except the blank control group were modeled by intraperitoneal injection with DEN(50 mg/kg)once a week for 16 consecutive weeks.At the same time,from the first week of modeling,the blank control group and the model group were administrated with 10 ml/kg water by gavage,and Zhenggan Prescription was administrated by gavage at corresponding doses once daily continuously until the 16th week.After 16 h of fasting without water,the rats were weighed and anaesthetized.Blood samples were collected from the heart,and the liver and thymus were weighed.The liver function indicators including alanine aminotransferase(ALT),alkaline phosphatase(ALP),albumin(ALB),andγ-glutamyl transpeptidase(GGT)were measured by fully automated biochemical methods.The liver tissue was stained with hematoxylin-eosin for the observation of the pathological changes.Immunohistochemistry and Western blotting were employed to examine the positive expression of alpha fetoprotein(AFP).The expression levels of the proteins involved in the PTEN/PI3K/AKT signaling pathway in the liver tissue were determined by Western blotting.Results Compared with the blank control group,the modeling reduced the body weight,increased the liver index,decreased thymus index,and elevated the levels of liver function indicators except ALB.The hepatocytes in the model group showed different degrees of degeneration,necrosis,disordered arrangement,cellular pleomorphism and other signs of precancerous lesions.Moreover,the modeling up-regulated the protein levels of AFP,p-PI3K,and p-AKT and down-regulated the protein level of PTEN(P<0.01).Compared with the model group,medium and high(P<0.05)doses of Zhenggan Prescription reduced the liver index,increased the thymus index(P<0.05 or P<0.001),lowered the serum levels of ALT,ALP,and GGT,elevated the ALB level,and reduced the areas of degeneration and necrosis.Moreover,they down-regulated the protein levels of AFP,p-PI3K,and p-AKT(P<0.01 or P<0.05)and up-regulated the protein level of PTEN(P<0.01).Conclusion Zhenggan Prescription can improve liver function indicators,mitigate the precancerous lesions,improve immunity,up-regulated the protein level of PTEN,and down-regulated the expression levels of AFP,p-PI3K,p-AKT.It may inhibit the activation of PTEN/PI3K/AKT signaling pathway to suppress the progression of precancerous lesions in the liver.
作者
孙童
胡世平
杨大国
黄胜
吴广阳
冉云
SUN Tong;HU Shi-ping;YANG Da-guo;HUANG Sheng;WU Guang-yang;RAN Yun(Beijing University of Chinese Medicine,Beijing 100029;Shenzhen Hospital(Longgang),Beijing University of Chinese Medicine,Shenzhen Guangdong 518100;The Third People's Hospital of Shenzhen,Shenzhen Guangdong 518112;Medical School,Hubei Minzu University,Enshi Hubei 445000;Hubei Enshi College,Enshi Hubei 445000)
出处
《世界中西医结合杂志》
2023年第7期1278-1284,1314,共8页
World Journal of Integrated Traditional and Western Medicine
基金
深圳市科技计划项目(JCYJ20180302150218892)
国家自然科学基金(81973733)
深圳市科技计划项目(JCYJ20220530172804010)
深圳市科技计划项目(JSGG20220226090405009)。
