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摘 要 目的:研究双参饮对肝纤维化模型大鼠的肝功能和肝纤维化程度的改善作用及其机制。方法:取健康SD大鼠,以含40%四氯化碳的橄榄油溶液连续7周腹腔注射,建立肝纤维化模型。将造模成功的47只大鼠随机分为模型组、阳性对照组和双参饮低、中、高剂量组,各组分别为9、9、9、10、10只;另取10只健康SD大鼠作为正常组。从造模结束后次周開始,模型组与正常组大鼠灌胃水,阳性对照组[秋水仙碱0.2 mg/(kg·d)]和双参饮各剂量组[双参饮水煎液3、6、12 g/(kg·d),以总生药量计算]大鼠分别灌胃相应药液,连续给药28 d。采用赖氏分析法检测血清中肝功能指标[丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBIL)、白蛋白(ALB)、球蛋白(GLB)]水平,采用酶联免疫吸附法检测血清中肝纤维化指标[透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型胶原(Ⅲ-PC)、Ⅳ型胶原前体(Ⅳ-C)]水平;采用苏木精-伊红染色法和苦味酸酸性复红染色法观察大鼠肝组织病理学变化;采用分光光度法检测大鼠肝组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、羟脯氨酸(HYP)水平;采用免疫组化法检测大鼠肝组织中α-平滑肌肌动蛋白(α-SMA)表达水平。结果:与正常组比较,模型组大鼠血清中ALT、AST、TBIL、HA、LN、PC-Ⅲ、Ⅳ-C水平均显著升高,ALB水平显著降低(P<0.05);大鼠肝组织出现空泡变性、纤维组织增生等病理现象;肝组织中SOD水平显著降低,MDA、HYP水平均显著升高(P<0.05);肝组织中可见大量α-SMA蛋白阳性染色颗粒,其蛋白表达水平显著升高(P<0.05)。与模型组比较,双参饮各剂量组大鼠血清中ALT、AST和肝纤维化指标水平均显著降低,ALB水平显著升高,双参饮中、高剂量组TBIL水平均显著降低(P<0.05);肝细胞变性、纤维化组织增生现象均有较大改善;肝组织中SOD水平显著升高,MDA、HYP水平均显著降低(P<0.05);α-SMA蛋白阳性染色明显减少,其蛋白表达水平显著降低(P<0.05)。结论:双参饮对肝纤维化模型大鼠有保肝、抗肝纤维化的作用,其机制可能主要是通过增强机体抗氧化能力、下调α-SMA蛋白表达、抑制肝星状细胞的活化、减少细胞外间质的合成,从而逆转肝纤维化进程。
关键词 双参饮;丹参;人参;肝纤维化;机制;大鼠
Study on Improvement Effect and Mechanism of Shuangshen Yin on Liver Fibrosis Model Rats
HUANG Yi1,ZHOU Gang2,ZHONG Shan3,ZHOU Zhi3,HU Xiaoyu1(1. Chengdu University of TCM, Chengdu 610075,China; 2. Bazhong Traditional Chinese Medicine Hospital, Sichuan Bazhong 636600, China; 3. Dept. of Infectious Disease,the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China)
ABSTRACT OBJECTIVE: To study improvement effect and mechanism of Shuangshen yin on liver function and the degree of liver fibrosis in liver fibrosis model rats. METHODS: Healthy SD rats were collected and given 40% CCl4 olive oil solution intraperitoneally for consecutive 7 weeks to induce liver fibrosis model. 47 model rats were randomly divided into model group, positive control group and Shuangshen yin low-dose, medium-dose and high-dose groups, with 9, 9, 9, 10, 10 rats respectively; another 10 healthy SD rats were included in normal group. From the next week after model induction, model group and normal group were given water intragastrically; positive control group [colchicines 0.2 mg/(kg·d)] and Shuangshenyin groups [Shuangshen yin water decoction 3, 6, 12 g/(kg·d), by crude drug] were given corresponding medicines respectively for consecutive 28 d. The serum levels of liver function indicators (ALT, AST, TBIL, ALB, GLB) were determined by Rye’s analysis method. The serum levels of liver fibrosis indexes (HA, LN, Ⅲ-PC, Ⅳ-C) were determined by ELISA. The pathological changes of liver tissues were observed by HE staining and VG staining. The levels of SOD, MDA and HYP in liver tissues were determined by spectrophotometry. The expression of α-SMA was detected by immunohistochemistry. RESULTS: Compared with normal group, the serum levels of ALT, AST, TBIL, HA, LN, PC-Ⅲ and Ⅳ-C in model group were increased significantly, and the level of ALB was decreased significantly (P<0.05). There were some pathological phenomena such as vacuolar degeneration and fibrous tissue proliferation in rat liver. The levels of SOD in liver tissue were decreased significantly, while the levels of MDA and HYP were increased significantly (P<0.05). A large number of positive staining granules of α-SMA protein were observed in liver tissue, and the expression level of α-SMA protein was significantly increased (P<0.05). Comparison of model group, the serum levels of ALT, AST and liver fibrosis indexes were significantly decreased and ALB level was significantly increased in Shuangshen yin groups, while the TBIL levels in Shuangshen yin medium-dose, high-dose groups were significantly decreased (P<0.05); the phenomena of hepatocyte degeneration and fibrosis tissue proliferation were significantly improved; the levels of SOD in liver tissue were significantly increased, and the levels of MDA and HYP were significantly decreased (P<0.05); the positive staining of α-SMA protein decreased significantly, and the expression level of α-SMA protein was decreased significantly (P<0.05). CONCLUSIONS: Shuangshen yin has protective and anti-fibrotic effects on liver in rats with hepatic fibrosis, the mechanism of which may be associated with enhancing antioxidant capacity, down-regulating the expression of α-SMA, inhibiting the activation of hepatic stellate cells, reducing extracellular matrix synthesis so as to reverse liver fibrosis. KEYWORDS Shuangshen yin; Salvia miltiorrhiza; Panax schinseng; Liver fibrosis; Mechanism; Rats
肝纖维化(Hepatic fibrosis)是指肝脏内弥漫性细胞外基质过度沉积,即肝脏纤维结缔组织大量增生,而其降解活性相对或绝对不足,使得大量细胞外基质沉积下来所形成[1]。它不是一个独立的疾病,而是许多慢性肝病的共同病理过程。抗肝纤维化治疗对于延缓慢性肝病的发展或逆转其病理过程具有重要的临床意义。
双参饮源于成都中医药大学附属医院感染科扈晓宇教授治疗肝纤维化的基础方。扈晓宇教授通过长期临床实践总结出肝纤维化的基本病机为正气亏虚、脉络瘀阻,而其所拟双参饮基础方由人参、丹参组成,利用这两种药材益气活血的功效治疗肝纤维化。前期临床研究已证实,该基础方具有益气扶正、活血化瘀的功效,对肝纤维化患者有较好的疗效[2]。但该方的作用机制尚不明确,故本研究拟通过考察肝纤维化模型大鼠采用双参饮水煎液干预治疗后的血清肝功能和肝纤维化指标,肝组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、羟脯氨酸(HYP)、α-平滑肌肌动蛋白(α-SMA)水平以及肝脏组织病理变化等,初步探究该方改善实验性肝纤维化的作用机制,为其在临床用于治疗肝纤维化提供理论依据。
1 材料
1.1 仪器
MS104S型电子天平[梅特勒-托利多国际贸易(上海)有限公司];Fresco 17型冷冻高速离心机(美国Bio-Rad Laboratories公司);KD-3368AM型全自动组织切片机(上海之信仪器有限公司);EVOS-FL型荧光显微镜(上海启文生物科技有限公司);756PC型紫外可见分光光度计(北京北拓仪器有限公司);JY-TSB型生物组织智能脱水机(湖北锦源医疗科技有限公司);TKY-KPA型病理组织烤片摊片机(湖北康泰医疗有限公司)。
1.2 药品与试剂
秋水仙碱片(阳性对照药物,云南植物药业有限公司,批号:H53020166,规格:0.5 mg);丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)测定试剂盒(上海纪宁科研试剂有限公司);总胆红素(TBIL)、白蛋白(ALB)、球蛋白(GLB)测定试剂盒(四川迈克生物科技股份有限公司);大鼠透明质酸(HA)酶联免疫吸附测定(ELISA)试剂盒、大鼠Ⅳ型胶原(Ⅳ-C)、大鼠层黏蛋白(LN)、大鼠Ⅲ型胶原前体(Ⅲ-PC)检测试剂盒(成都里来生物科技股份有限公司);SOD、MDA、HYP检测试剂盒(广州创美生物科技有限公司);兔α-SMA多克隆抗体(美国Abcam公司);NovoLink聚合物检测系统RE7280-K检测试剂盒(德国Leica公司);四氯化碳(CCl4,分析纯,成都金山化学试剂有限公司;使用时以橄榄油配成40%的油溶液);苏木精-伊红(HE)染色试剂盒、苦味酸酸性复红(VG)染色试剂盒(上海歌凡生物科技有限公司);橄榄油为市售食用橄榄油;水为高压灭菌水。
1.3 双参饮水煎液
双参饮水煎液由四川省中医院煎药房煎制(方中人参-丹参的质量比为1 ∶ 3,药材均购自成都中医药大学附属医院中药房)。煎煮方法:按处方比例称取人参、丹参药材,加入6倍量水(mL/g)浸泡,每隔10 min观察浸润程度,直至药材浸透;煎煮3次,每次1 h;合并滤液,静置,取上清液浓缩制成相当于含生药总量1.2 g/mL的浓缩液,低温保存,待用。
1.4 动物
SFP级SD雄性大鼠,65只,鼠龄10周,体质量为(200±20)g,购自成都里来生物科技有限公司,动物生产许可证号:SCXK(川)2016-011。大鼠均适应性喂养1周,观察无异常后进行后续实验。所有大鼠全程均给予普通饲料和水喂养。
2 方法
2.1 大鼠分组、造模和给药
取大鼠65只,通过随机数字表法抽取10只纳入正常组,其余大鼠均纳入造模组。参考文献[3-4],本实验以CCl4建立大鼠肝纤维化模型:造模组大鼠腹腔注射含40%CCl4的橄榄油溶液,首剂3 mL/kg,之后每次1.5 mL/kg,每周2次(间隔为3~4 d),连续注射7周。造模结束后,有5只大鼠死亡,在剩余50只造模组大鼠中随机抽取3只,以10 mg/mL戊巴比妥溶液麻醉后断颈处死,剖取其肝脏组织,制作HE染色切片和VG染色切片,于显微镜下观察造模是否成功。参考2006年《肝纤维化中西医结合诊疗指南》中的纤维化程度分期标准[5](S0期:无纤维化;S1期:汇管区纤维化扩大,局限窦周及小叶内纤维化;S2期:汇管区周围纤维化,纤维间隔形成,小叶结构保留;S3期:纤维间隔伴小叶结构紊乱,无肝硬化;S4期:早期肝硬化)进行判断:当镜下观察结果符合肝纤维化病理变化特征且分期为S2~S4期时则视为造模成功。剩余47只造模组大鼠通过随机数字表法分为模型组、阳性对照组[秋水仙碱0.2 mg/(kg·d);剂量按成人临床用量换算制定]和双参饮低、中、高剂量组[双参饮水煎液3、6、12 g/(kg·d),以总生药量计算;剂量按成人临床用量的2.5、5、10倍换算制定],各组分别为9、9、9、10、10只。从第8周(即造模结束后次周)开始,模型组与正常组大鼠灌胃水,阳性对照组和双参饮各剂量组大鼠灌胃相应药液(秋水仙碱片以水配制为0.02 mg/mL的药液;双参饮水煎液以水稀释为含总生药量0.3、0.6、1.2 g/mL的药液),给药体积均为10 mL/kg,连续给药28 d。
2.2 大鼠血清肝功能和肝纤维化指标检测
给药结束后,全部大鼠禁食不禁水24 h,然后以10 mg/mL的戊巴比妥溶液腹腔注射麻醉,立即经心脏采血,静置凝固后取上层血清。按相应试剂盒说明书操作,采用赖氏分析法[6]检测血清中肝功能指标(ALT、AST、TBIL、ALB、GLB)水平,采用ELISA法检测血清中肝纤维化指标(HA、LN、Ⅲ-PC、Ⅳ-C)水平。
关键词 双参饮;丹参;人参;肝纤维化;机制;大鼠
Study on Improvement Effect and Mechanism of Shuangshen Yin on Liver Fibrosis Model Rats
HUANG Yi1,ZHOU Gang2,ZHONG Shan3,ZHOU Zhi3,HU Xiaoyu1(1. Chengdu University of TCM, Chengdu 610075,China; 2. Bazhong Traditional Chinese Medicine Hospital, Sichuan Bazhong 636600, China; 3. Dept. of Infectious Disease,the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China)
ABSTRACT OBJECTIVE: To study improvement effect and mechanism of Shuangshen yin on liver function and the degree of liver fibrosis in liver fibrosis model rats. METHODS: Healthy SD rats were collected and given 40% CCl4 olive oil solution intraperitoneally for consecutive 7 weeks to induce liver fibrosis model. 47 model rats were randomly divided into model group, positive control group and Shuangshen yin low-dose, medium-dose and high-dose groups, with 9, 9, 9, 10, 10 rats respectively; another 10 healthy SD rats were included in normal group. From the next week after model induction, model group and normal group were given water intragastrically; positive control group [colchicines 0.2 mg/(kg·d)] and Shuangshenyin groups [Shuangshen yin water decoction 3, 6, 12 g/(kg·d), by crude drug] were given corresponding medicines respectively for consecutive 28 d. The serum levels of liver function indicators (ALT, AST, TBIL, ALB, GLB) were determined by Rye’s analysis method. The serum levels of liver fibrosis indexes (HA, LN, Ⅲ-PC, Ⅳ-C) were determined by ELISA. The pathological changes of liver tissues were observed by HE staining and VG staining. The levels of SOD, MDA and HYP in liver tissues were determined by spectrophotometry. The expression of α-SMA was detected by immunohistochemistry. RESULTS: Compared with normal group, the serum levels of ALT, AST, TBIL, HA, LN, PC-Ⅲ and Ⅳ-C in model group were increased significantly, and the level of ALB was decreased significantly (P<0.05). There were some pathological phenomena such as vacuolar degeneration and fibrous tissue proliferation in rat liver. The levels of SOD in liver tissue were decreased significantly, while the levels of MDA and HYP were increased significantly (P<0.05). A large number of positive staining granules of α-SMA protein were observed in liver tissue, and the expression level of α-SMA protein was significantly increased (P<0.05). Comparison of model group, the serum levels of ALT, AST and liver fibrosis indexes were significantly decreased and ALB level was significantly increased in Shuangshen yin groups, while the TBIL levels in Shuangshen yin medium-dose, high-dose groups were significantly decreased (P<0.05); the phenomena of hepatocyte degeneration and fibrosis tissue proliferation were significantly improved; the levels of SOD in liver tissue were significantly increased, and the levels of MDA and HYP were significantly decreased (P<0.05); the positive staining of α-SMA protein decreased significantly, and the expression level of α-SMA protein was decreased significantly (P<0.05). CONCLUSIONS: Shuangshen yin has protective and anti-fibrotic effects on liver in rats with hepatic fibrosis, the mechanism of which may be associated with enhancing antioxidant capacity, down-regulating the expression of α-SMA, inhibiting the activation of hepatic stellate cells, reducing extracellular matrix synthesis so as to reverse liver fibrosis. KEYWORDS Shuangshen yin; Salvia miltiorrhiza; Panax schinseng; Liver fibrosis; Mechanism; Rats
肝纖维化(Hepatic fibrosis)是指肝脏内弥漫性细胞外基质过度沉积,即肝脏纤维结缔组织大量增生,而其降解活性相对或绝对不足,使得大量细胞外基质沉积下来所形成[1]。它不是一个独立的疾病,而是许多慢性肝病的共同病理过程。抗肝纤维化治疗对于延缓慢性肝病的发展或逆转其病理过程具有重要的临床意义。
双参饮源于成都中医药大学附属医院感染科扈晓宇教授治疗肝纤维化的基础方。扈晓宇教授通过长期临床实践总结出肝纤维化的基本病机为正气亏虚、脉络瘀阻,而其所拟双参饮基础方由人参、丹参组成,利用这两种药材益气活血的功效治疗肝纤维化。前期临床研究已证实,该基础方具有益气扶正、活血化瘀的功效,对肝纤维化患者有较好的疗效[2]。但该方的作用机制尚不明确,故本研究拟通过考察肝纤维化模型大鼠采用双参饮水煎液干预治疗后的血清肝功能和肝纤维化指标,肝组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、羟脯氨酸(HYP)、α-平滑肌肌动蛋白(α-SMA)水平以及肝脏组织病理变化等,初步探究该方改善实验性肝纤维化的作用机制,为其在临床用于治疗肝纤维化提供理论依据。
1 材料
1.1 仪器
MS104S型电子天平[梅特勒-托利多国际贸易(上海)有限公司];Fresco 17型冷冻高速离心机(美国Bio-Rad Laboratories公司);KD-3368AM型全自动组织切片机(上海之信仪器有限公司);EVOS-FL型荧光显微镜(上海启文生物科技有限公司);756PC型紫外可见分光光度计(北京北拓仪器有限公司);JY-TSB型生物组织智能脱水机(湖北锦源医疗科技有限公司);TKY-KPA型病理组织烤片摊片机(湖北康泰医疗有限公司)。
1.2 药品与试剂
秋水仙碱片(阳性对照药物,云南植物药业有限公司,批号:H53020166,规格:0.5 mg);丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)测定试剂盒(上海纪宁科研试剂有限公司);总胆红素(TBIL)、白蛋白(ALB)、球蛋白(GLB)测定试剂盒(四川迈克生物科技股份有限公司);大鼠透明质酸(HA)酶联免疫吸附测定(ELISA)试剂盒、大鼠Ⅳ型胶原(Ⅳ-C)、大鼠层黏蛋白(LN)、大鼠Ⅲ型胶原前体(Ⅲ-PC)检测试剂盒(成都里来生物科技股份有限公司);SOD、MDA、HYP检测试剂盒(广州创美生物科技有限公司);兔α-SMA多克隆抗体(美国Abcam公司);NovoLink聚合物检测系统RE7280-K检测试剂盒(德国Leica公司);四氯化碳(CCl4,分析纯,成都金山化学试剂有限公司;使用时以橄榄油配成40%的油溶液);苏木精-伊红(HE)染色试剂盒、苦味酸酸性复红(VG)染色试剂盒(上海歌凡生物科技有限公司);橄榄油为市售食用橄榄油;水为高压灭菌水。
1.3 双参饮水煎液
双参饮水煎液由四川省中医院煎药房煎制(方中人参-丹参的质量比为1 ∶ 3,药材均购自成都中医药大学附属医院中药房)。煎煮方法:按处方比例称取人参、丹参药材,加入6倍量水(mL/g)浸泡,每隔10 min观察浸润程度,直至药材浸透;煎煮3次,每次1 h;合并滤液,静置,取上清液浓缩制成相当于含生药总量1.2 g/mL的浓缩液,低温保存,待用。
1.4 动物
SFP级SD雄性大鼠,65只,鼠龄10周,体质量为(200±20)g,购自成都里来生物科技有限公司,动物生产许可证号:SCXK(川)2016-011。大鼠均适应性喂养1周,观察无异常后进行后续实验。所有大鼠全程均给予普通饲料和水喂养。
2 方法
2.1 大鼠分组、造模和给药
取大鼠65只,通过随机数字表法抽取10只纳入正常组,其余大鼠均纳入造模组。参考文献[3-4],本实验以CCl4建立大鼠肝纤维化模型:造模组大鼠腹腔注射含40%CCl4的橄榄油溶液,首剂3 mL/kg,之后每次1.5 mL/kg,每周2次(间隔为3~4 d),连续注射7周。造模结束后,有5只大鼠死亡,在剩余50只造模组大鼠中随机抽取3只,以10 mg/mL戊巴比妥溶液麻醉后断颈处死,剖取其肝脏组织,制作HE染色切片和VG染色切片,于显微镜下观察造模是否成功。参考2006年《肝纤维化中西医结合诊疗指南》中的纤维化程度分期标准[5](S0期:无纤维化;S1期:汇管区纤维化扩大,局限窦周及小叶内纤维化;S2期:汇管区周围纤维化,纤维间隔形成,小叶结构保留;S3期:纤维间隔伴小叶结构紊乱,无肝硬化;S4期:早期肝硬化)进行判断:当镜下观察结果符合肝纤维化病理变化特征且分期为S2~S4期时则视为造模成功。剩余47只造模组大鼠通过随机数字表法分为模型组、阳性对照组[秋水仙碱0.2 mg/(kg·d);剂量按成人临床用量换算制定]和双参饮低、中、高剂量组[双参饮水煎液3、6、12 g/(kg·d),以总生药量计算;剂量按成人临床用量的2.5、5、10倍换算制定],各组分别为9、9、9、10、10只。从第8周(即造模结束后次周)开始,模型组与正常组大鼠灌胃水,阳性对照组和双参饮各剂量组大鼠灌胃相应药液(秋水仙碱片以水配制为0.02 mg/mL的药液;双参饮水煎液以水稀释为含总生药量0.3、0.6、1.2 g/mL的药液),给药体积均为10 mL/kg,连续给药28 d。
2.2 大鼠血清肝功能和肝纤维化指标检测
给药结束后,全部大鼠禁食不禁水24 h,然后以10 mg/mL的戊巴比妥溶液腹腔注射麻醉,立即经心脏采血,静置凝固后取上层血清。按相应试剂盒说明书操作,采用赖氏分析法[6]检测血清中肝功能指标(ALT、AST、TBIL、ALB、GLB)水平,采用ELISA法检测血清中肝纤维化指标(HA、LN、Ⅲ-PC、Ⅳ-C)水平。