葛根芩连汤及其有效组分干预非酒精性脂肪性肝炎PPARγ的作用机制研究
摘要
本文通过文献综述、实验研究两方面阐述了现代医学和中医药对非酒精性脂肪性肝炎(NASH)的认识、临床与实验研究现状,探讨了葛根芩连汤及其有效组分干预非酒精性脂肪性肝炎PPARγ的作用机制研究,以达到探索古方新用,提升经方在该领域研究价值和临床治疗水平,同时为开发防治NASH有效的中药制剂提供新思路和实验依据。
1文献综述
通过近10年的国内外文献资料分析,归纳了NASH现代医学的病因、发病机制、治疗研究近况,中医药关于NASH病因病机、证候学、临床研究及NASH实验相关模型的研究进展。
2葛根芩连汤对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎实验研究
目的
探讨葛根芩连汤对NASH大鼠模型的药效学作用及机制。
方法
以高脂饮食建立大鼠NASH模型,并于造模同时进行药物干预；实验第8周末进行血清ALT、AST、TG、HDL-C、LDL-C、TNF-a、IL-6、IL-10及肝组织TNF-a、IL-6、IL-10的测定,并进行肝组织PPARγ Western-blot与PCR检测,同时油红O、HE染色观察肝脏病理切片。
结果
(1)一般情况及病理形态学：模型组大鼠毛色枯黄黯淡,喜团聚蜷卧,饮食量较空白组减少,大便干稀不调,体重也随着造模时间逐渐增加,NAS组织学病理评分总分>5分(P<0.05)；葛根芩连汤高、低剂量组脂肪变性、小叶内炎症、气球样变均明显下降,有显著性差异；葛根芩连汤高、低剂量组较模型组病理总分均显著下降<5分(P<0.05)。
(2)肝脏酶学：模型组血清ALT、AST水平均明显高于空白组,有显著统计学差异(P<0.05),葛根芩连汤高、低剂量组血清ALT、AST水平均较模型组显著降低(P<0.05)。
(3)血脂及肝脂：与空白组相比,模型组血清HDL显著降低,血清LDL、TG、TC明显增加(P<0.05)。葛根芩连汤高剂量组血清HDL水平与模型组相比增加明显(P<0.05)；葛根芩连汤低剂量组与模型组相比,血清LDL水平下降明显(P<0.05)；与模型组相比,葛根芩连汤高、低剂量组血清TC均降低明显(P<0.05)；对于血清甘油三酯,各给药组与模型组之间无统计学意义(P>0.05)。
(4)血清及肝组织IL-6、IL-10、TNF-a水平：与空白组相比,模型组血清及肝组织IL-10显著降低,血清IL-6、TNF-α明显增加(P<0.05)。与模型组相比,葛根芩连汤高、低剂量组可明显增加血清及肝组织IL-10含量,降低血清及肝组织IL-6、TNF-α水平(P (5)肝组织PPARγ蛋白及mRNA表达：与空白组相比,模型组肝组织PPARγ蛋白表达及mRNA水平均降低(P<0.05)；葛根芩连汤高、低剂量组与模型组相比,可明显改善肝组织PPAR γ蛋白表达及mRNA表达(P<0.05)。
结论
葛根芩连汤高、低剂量均能显著改善高脂饮食饲喂诱导的NASH大鼠肝组织病理,改善脂肪变、炎性因子浸润；能够降低血清转氨酶水平、改善血脂代谢紊乱；能够通过抑制促炎因子、提高抑炎因子两方面动态调控炎症的发展；同时通过对肝组织PPAR γ的调控达到干预大鼠NASH的作用。
3游离脂肪酸诱导NASH体外模型的建立及动态监测
目的
研究游离脂肪酸诱导的非酒精性脂肪性肝炎体外模型,动态观测细胞脂肪变性、炎症、过氧化损伤及糖转运缺陷发生发展的特点。
方法
以HepG2细胞为实验对象,采用不同浓度游离脂肪酸(250μM,500μ M,1000μM)对细胞进行不同时间(12h,18h,24h)的干预刺激,并通过油红0染色,酶联免疫的方法分别对细胞内脂肪变及细胞培养上清液中IL-8, TNF-a, SOD, MDA, GLUT4进行测定评估。
结果
FFA作用的实验各组,随着时间的延长及浓度的增加,细胞脂肪样变、IL-8、TNF-a、SOD、MDA及GLUT4均有不同程度的改变,以24h0.5mM FFA、24h1mM FFA最为明显,与同期空白对照组相比,其在IL-8, TNF-a、SOD、MDA、GLUT4的表达方面均有统计学意义(P<0.05),油红0染色可见典型的“印戒”状。
结论
应用游离脂肪酸诱导建立体外NASH模型成模效果好,重复率高,并提示500μM FFA24h为佳。
4葛根芩连汤对NASH细胞模型的干预研究
目的
探讨葛根芩连汤对非酒精性脂肪性肝炎体外模型的影响。
方法
用500μmol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的葛根芩连汤干预NASH体外模型,MTT法检测葛根芩连汤的安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-a、IL-8及GLUT4。
结果
(1)NASH细胞模型组培养液中TNF-α及IL-8含量明显高于空白对照组(P<0.01),GLUT4含量明显低于空白对照组。
(2)NASH模型组中同时加入葛根芩连汤可明显改善细胞脂肪样变,减少TNF-αIL-8分泌,提高GLUT4含量。
结论
葛根芩连汤能够通过影响TNF-α、IL-8及GLUT4改善NASH体外模型炎症及糖脂代谢。
5葛根素、小檗碱联合用药干预NASH细胞模型的的实验研究
目的
探讨葛根素、小檗碱联合用药干预非酒精性脂肪性肝炎体外模型的相关剂量及作用效果。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的葛根素、小檗碱单药及组合用药干预NASH体外模型。MTT法测定两种单体对非酒精性脂肪性肝病体外模型细胞安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-α、IL-8及GLUT4水平。
结果
(1)500μ mol/LFFA作用NASH模型组,可见细胞胞浆内被染成红色的颗粒状脂滴,并呈“印戒”状形态。不同浓度葛根素、小檗碱单用及联合用药(p500、p1000、p2000、b25、b50、b25p1000、b50p500,单位u mol/L)干预组,可见细胞内红色脂滴有所减少,单体联合用药组优于同浓度单独用药组,基本达到以小剂量联合不次于高剂量单独用药的目的。
(2)500μ mol/L FFA刺激24h后的HepG2细胞,其细胞培养上清液中炎性因子TNF-α及IL-8含量明显增加,GLUT4含量明显下降,与空白组比,差异显著(P<0.05)。
随着小檗碱、葛根素单独使用浓度的增加,IL-8水平较模型组明显降低,以b50、b100、p2000差异明显(P<0.05)；组合用药后b25p1000、b50p500均较模型组有统计学意义(P<0.05),b25p1000较b25效果明显(P<0.05),b50p500较p500、p1000效果明显(P<0.05)。
对于TNF-a,随着小檗碱、葛根素单独使用浓度的增加,其上清液含量显著降低,均较模型组用统计学差异(P<0.05)；组合用药后,TNF-a含量的变化优于单独使用时,b25p1000较p1000、p2000效果明显(P<0.05),b50p500较b50效果明显(P<0.05)。
对于GLUT4,随着小檗碱、葛根素单独使用浓度的增加,其上清液含量显著增加,以b50、b100、p500、p1000、p2000较模型组用统计学差异(P<0.05),且葛根素对于增加GLUT4含量作用优于小檗碱；组合用药后,GLUT4含量的变化优于小檗碱单独使用,b25p1000在加入葛根素后较b25、b50效果明显增强,b50p500较b50、b100效果明显增加(P<0.05)。
结论
葛根素、小檗碱组合用药在改善细胞病理、TNF-α、IL-8、GLUT4水平方面存在协同作用,其组合用药效果较单独用药效果增强。体外实验中,基本能够确定在一定范围内,小檗碱与葛根素组合比例在1：40-1：10为佳。
6葛根素、小檗碱、黄芩苷联合用药干预NASH细胞模型的实验研究
目的
探讨葛根素、小檗碱、黄芩苷联合用药干预非酒精性脂肪性肝炎体外模型的相关剂量及作用效果。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的黄芩苷单药及葛根素、小檗碱、黄芩苷组合用药干预NASH体外模型。MTT法测定两种单体对非酒精性脂肪性肝病体外模型细胞安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-α及IL-8水平。
结果
(1)500μ mol/L FFA作用NASH模型组,可见细胞胞浆内被染成红色的颗粒状脂滴,并呈“印戒”状形态。不同浓度黄芩苷单用及葛根素、小檗碱、黄芩苷联合用药(bai25、bai50、bai100、bai200、b25p1000bai25、b25p1000bai50、b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai,单位100μ mol/L)干预组,可见细胞内红色脂滴有所减少,单体联合用药组效果明显。
(2)500μ mol/L FFA刺激24h后的HepG2细胞,其细胞培养上清液中炎性因子TNF-α及工L-8含量明显增加,与空白组比差异显著(P，0.05)。
随着黄芩苷单独使用浓度的增加,IL-8水平较模型组明显降低,以bai200差异明显(P<0.05)；组合用药后b25p1000bai25、b25p1000bai50、b25p1000bai100、b50p500bai25、 b50p500bai50、b50p500bai100均较模型组有统计学意义(P，0.05),在各比例组合中,以b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai100效果最为明显(P<0.05)。
对于TNF-a,随着黄芩苷单独使用浓度的增加,其上清液含量显著降低,均较模型组用统计学差异(P<0.05);组合用药后,b25p1000bai25、b25pl000bai50、b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai100均较模型组有统计学意义(P<0.05),且效果优于黄芩苷单独使用时。在各比例组合中,以b50p500bai50、b50p500bai100效果最为明显(P 结论
葛根素、小檗碱、黄芩苷联合用药(b25p1000bai25、b25p1000bai50、b25p1000bai100、 b50p500bai25、b50p500bai50、b50p500bai100)在减少细胞内红色脂滴方面,存在显著协同作用；对于TNF-α、IL-8,通过单体组合使用,可在降低黄芩苷浓度的前提下保证干预效果。体外实验中,基本能够确定在一定范围内,小檗碱、葛根素、黄芩苷组合比例在1：10：1-1：10：2为佳。
7PPAR γ与炎性因子在NASH细胞模型中的相关性研究
目的
明确PPAR y与炎性因子在NASH细胞模型中的关系。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；造模同时给与不同浓度GW9662／罗格列酮刺激；Elisa法检测细胞培养上清液中TNF-α及IL-8水平,油红0染色测定细胞内脂滴。
结果
(1)与模型组比较,GW966210u M加入后,IL-8及TNF-a含量均增加,但未出现统计学意义。加入罗格列酮2μM后,IL-8及TNF-α含量显著降低,与模型组、gw10组相比,均具有统计学差异(P<0.05)。各单体与gw10组合中,IL-8及TNF-α含量均降低,与模型组、gw10组比较差异显著(P<0.05)。
为了避免因模型带来的非GW9662因素造成的工L-8及TNF-α含量增加,补充了GW9662对未加入FFA诱导的“正常”细胞的作用。结果显示,不同浓度的GW9662均使“正常”细胞上清液中IL-8及TNF-α含量增加,与未加入GW9662组比,差异均具有统计学意义(P<0.05)。
(2)读取OD值进行细胞内脂滴含量分析,GW9662不同浓度较模型组比,脂滴含量均升高,差异显著,具有统计学意义(P<0.05)。
结论
PPAR y阻断剂GW9662可以通过阻断PPAR y促进炎症发展；葛根素、小檗碱、黄芩苷具有潜在的PPAR γ激动剂的作用；PPAR γ与脂质沉积代谢密切相关。
8葛根芩连汤有效组分对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎药效学观察
目的
明确葛根芩连汤有效组分(葛根素、小檗碱、黄芩苷)对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎药效学作用。
方法
以高脂饮食建立大鼠NASH模型,并于造模同时进行药物干预(通过正交表确定组数)；实验第8周末进行血清ALT、AST、TG、HDL-C、LDL-C的测定,同时油红O、HE染色观察肝脏病理切片。
结果
(1)一般情况及病理形态学：模型组大鼠毛色黯淡枯黄,喜团聚蜷卧,饮食量较空白组减少,大便干稀不调,模型组肝组织HE染色存在明显的肝细胞脂肪变性,肝小叶界限不清,肝索结构紊乱,大量空泡及气球样变产生,中性粒细胞浸润,油红0染色可见大量红色脂滴,颜色较深,显示肝组织内脂滴弥漫、含量较多,肝小叶内脂滴融合成片；葛根芩连汤高、低剂量组HE染色病变有不同程度减轻,损伤面积明显减小,胞内炎性细胞浸润减少,空泡及气球样变减少,细胞及小叶结构不同程度恢复,油红0染色脂滴减少。
(2)肝脏酶学：模型组血清ALT、AST均明显高于空白组,有显著差异(P<0.01),各给药组血清ALT、AST水平均较模型组均显著降低(P<0.01),其中F、G、H组在改善血清ALT、AST方面效果明显优于HH组(P<0.01)；D、E组仅在改善AST方面优于HH组(P<0.01)
(3)血脂及肝脂：与空白组相比,模型组HDL显著降低,LDL、TG、TC显著增高,具有统计学差异(P0.05)。对于血清总胆固醇,A、B、C、D、E、F、G、HH组与模型组之间均差异显著(P0.05)。
结论
葛根素为主药的单体组合,对肝组织病理改善,特别是脂滴改善最为明显；以抗炎类小檗碱、黄芩苷为主药的组合,对转氨酶影响最大；不同比例单体组合对血脂的调节都具有积极作用,但随着各单体所占比例的变化,并未见明确的规律性,一些组合的效能基本与葛根芩连汤高剂量组相当,提示组合明确的单体配伍可以在某些方面达到中药原方的效果。
Comprehensive learning of non-alcoholic steatohepatitis (NASH) in both modern medicine and traditional Chinese medicine (TCM) is demonstrated through literature review and experimental study in this paper. Efficacy and mechanism of GeGenQinLian Decoction and its componets are explored in in vivo and in vitro study. The research provided a basic reveal of the mechanism of GeGenQinLian Decoction and its componets, which may give a great contribution to clinical therapeutic levels and new grug discovery.
1Literature review
Through literature analysis in recent10years both at home and abroad, the etiology, pathogenesis, and treatment of modern medicine, also etiology and pathogenesis, syndrome differentiation, physical sciencetreatment progress of TCM have been review.
2Effects and Mechinsm of GeGenQinLian Decoction in NASH Rats induced by High-Fat Diet
Objective
To investigate the efficacy and mechinsm of GeGenQinLian Decoction on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.
Method
The SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by GeGenQinLian Decoction in high and low doses. The change of serum transaminase, serum lipid, serum inflammation factors, liver inflammation factors and the liver pathology had been tested to observe the efficacy. Meanwhile, PPAR γ protein and mRNA in liver were also detected.
Outcome
(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups, together with significantly reduced NAS score. And there was no significant difference between treatment groups.
(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased. The serum ALT and AST decreased both in high and low dose of GeGenQinLian Decoction group.
(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group. The high dose groups of GeGenQinLian Decoction increased the serum HDL-C compared with the model group, while the low dose of GeGenQinLian Decoction decreased the serum LDL-C significantly. Both the high and low dose of GeGenQinLian Decoction decreased serum TC.
(4) Serum and liver IL-6, IL-10and TNF-α:Compared with blank control group, the serum and liver IL-6and TNF-α in model group were obviously increased, while the serum and liver IL-10decrease. GeGenQinLian Decoction can significantly increase the leverl of serum and liver IL-10and decrease the serum and liver IL-6and TNF-α.
(5) liver PPAR γ protein and mRNA expression:Compared with blank control group, the liver PPAR γ protein and mRNA expressions were both declined, which were increased in GeGenQinLian Decoction groups.
Conclusion
GeGenQinLian Decoction can significantly improve liver pathology of the high-fat diet-induced NASH rats; improving steatosis and infiltration of inflammatory cytokines; reducing serum transaminase levels;improving dyslipidemia; and achieving intervention on NASH rats by PPAR γ regulation.
3A Cell Model of Nonalcoholic Steatohepatitis Induced by FFA:Establishment and Dynamic Monitoring
Objective
To establish a cell model of nonalcoholic steatohepatitis (NASH) by free fatty acid (FFA) and to dynamically monitor the pathological features of steatosis, oxidative damage and inflammation in these cells.
Methods
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. For the treatment, palmitic and oleic acid0.1M stock solutions were prepare by dissolving FFA in DMSO (with a final concentration less than0.1%). The cells were exposed to increasing concentrations of a fresh mixture of exogenous FFA (250μmol/L,500μmol/L,1000μmol/L) in molar ratio1:2palmitic:oleic at different time points (12h,18,24h) respectively. Oil red0staining and the IL-8, TNF-α, SOD, MDA, GLUT4 in supernatant were evaluated.
Outcome
Steatosis could be seen in FFA groups to different extents. IL-8, TNF-α, SOD, MDA and GLUT4gradually differed according to the change of stimulation time by different FFA concentrations, especially in the groups with0.5mM and1mM FFA concentrations for24h, of which groups there could be seen a typical steatosis.
Conclusion
A cell model of NASH has been created successfully by FFA.
4Research of Gegenqinlian Decoction on NASH in vitro
Objective
Free fatty acid induced NASH model was used to evaluate the effects of Gegenqinlian Decoction.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of Gegenqinlian Decoction were meanwhile added. MTT, Oil red0staining and the IL-8, TNF-α, GLUT4in supernatant were evaluated.
Outcome
(1) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group (P<0.01).
(2) Gegenqinlian Decoction can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content.
Conclusion
Gegenqinlian Decoction can improve lipid metabolism and inflammation through influencing TNF-a, IL-8and GLUT4.
5Experimental Studies of the Combination Effects of Berberine and Puerarin on NASH in vitro
Objective
To discuss the combination effects of puerarin and berberine on NASH in vi tro.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μ g/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μ mol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin and berberine combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8, TNF-a, GLUT4in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, different concentrations of puerarin and berberine alone and combination (p500, p1000, p2000, b25, b50, b25p1000, b50p500, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.
(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group(P<0.01). Different concentrations of puerarin and berberine combination can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content(P<0.01).
Conclusion
Puerarin and berberine combination can improve cellular pathology, influencing the levels of TNF-a, IL-8and GLUT4, which showed a synergistic effect and better than a single drug effect.
6Experimental Studies of the Combination Effects of Berberine, Puerarin and baicalin on NASH in vitro
Objective
To discuss the combination effects of puerarin, berberine and baicalin on NASH in vitro.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin, berberine and baicalin combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8and TNF-a in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, different concentrations of puerarin, berberine and baicalin alone and combination (bai25, bai50, bai100, bai200, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.
(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01). Different concentrations of puerarin, berberine and baicalin combination can significantly improve cell degeneration and steatosis and reduce TNF-a, IL-8secretion (P<0.01).
Conclusion
Puerarin, berberine, and baicalin combination can improve cellular pathology, influencing the levels of TNF-a and IL-8, which showed a synergistic effect. The better proportion of berberine, puerarin, baicalin combination was1:10:1-1:10:2in vitro.
7The Related Studies between PPAR γ and Inflammatory Factors on NASH in vitro
Objective
To clarify the relationship between PPAR γ and inflammatory factors.
Method
NASH model in vitro was established using500μmol/L FFA for-24h. The agonists of PPAR γ Rosiglitazone and antaonist of PPAR γ GW9662were meanwhile added.Oil red0staining and the IL-8and TNF-a in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, the GW966210μmol/L group can increase IL-8and TNF-a level; while IL-8and TNF-a were significantly reduced after adding rosiglitazone2μmol/L (P<0.05). In the groups of GW966210μmol/L plus with each monomer, IL-8and TNF-a levels were lowerd compared with the model group and GW966210μmol/L group (P<0.05).
In order to avoid an increase caused by model, GW9662was added to the "normal" cells without FFA. The results showed that different concentrations of GW9662increased IL-8and TNF-a levels compared with the group without GW9662(P<0.05).
(2) The intracellular lipid droplets content was analysized by reading OD values, lipid droplets levels were increased significantly in groups with different concentrations GW9662compared with the model group (P<0.05).
Conclusion
PPAR γ antagonist GW9662can promote inflammation by blocking PPAR γ; puerarin, berberine and baicalin hold the potential effects as PPAR γ agonist; PPAR γ is closely related to lipid metabolism.
8Effects of Components in GeGenQinLian Decoction in NASH Rats Induced by High-Fat Diet
Objective
To investigate the efficacy of components in GeGenQinLian Decoction (puerarin, berberine, baicalin) on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.
Method
The SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by the componts of GeGenQinLian Decoction (number of groups identified by orthogonal table). The change of serum transaminase, serum lipid and the liver pathology had been tested to observe the efficacy.
Outcome
(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups. And there was no significant difference between the all treatment groups.
(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased (P<0.01). The serum ALT and AST decreased both in all treatment groups, in which F, G, H groups were better than HH gtoup in inproving ALT and AST, and D, E group were better than HH group in inproving AST (P<0.01).
(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group (P<0.01). The A, B, D, E, I, HH groups increased the serum HDL-C compared with the model group, in which HH group was better than B, E groups (P<0.01). Compared with the model group, each treatment groups decreased serum LDL levels significantly (P<0.01), in which the HH group was better than E, F, H, I groups (P<0.01). For the serum triglycerides, the level in A, C, D, E, F, G, HH groups decreased compared with model group (P<0.01). For the serum total cholesterol, the level in A, B, C, D, E, F, G, HH group decreased compared with model group (P<0.01).
Conclusion
GeGenQinLian Decoction can significantly improve of the high-fat diet-induced NASH rats and achieve intervention on NASH rats by PPAR γ regulation.
Different proportions of monomer composition gave positive effects to the liver pathology, serum transaminase levels and dyslipidemia. With some certain propotion, it showd an equal effect to the high dose of GeGenQinLian Decoction, suggesting a clear monomer combination could achieve the effect at some aspects with the original formula in traditional Chinese medicine.
1文献综述
通过近10年的国内外文献资料分析,归纳了NASH现代医学的病因、发病机制、治疗研究近况,中医药关于NASH病因病机、证候学、临床研究及NASH实验相关模型的研究进展。
2葛根芩连汤对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎实验研究
目的
探讨葛根芩连汤对NASH大鼠模型的药效学作用及机制。
方法
以高脂饮食建立大鼠NASH模型,并于造模同时进行药物干预；实验第8周末进行血清ALT、AST、TG、HDL-C、LDL-C、TNF-a、IL-6、IL-10及肝组织TNF-a、IL-6、IL-10的测定,并进行肝组织PPARγ Western-blot与PCR检测,同时油红O、HE染色观察肝脏病理切片。
结果
(1)一般情况及病理形态学：模型组大鼠毛色枯黄黯淡,喜团聚蜷卧,饮食量较空白组减少,大便干稀不调,体重也随着造模时间逐渐增加,NAS组织学病理评分总分>5分(P<0.05)；葛根芩连汤高、低剂量组脂肪变性、小叶内炎症、气球样变均明显下降,有显著性差异；葛根芩连汤高、低剂量组较模型组病理总分均显著下降<5分(P<0.05)。
(2)肝脏酶学：模型组血清ALT、AST水平均明显高于空白组,有显著统计学差异(P<0.05),葛根芩连汤高、低剂量组血清ALT、AST水平均较模型组显著降低(P<0.05)。
(3)血脂及肝脂：与空白组相比,模型组血清HDL显著降低,血清LDL、TG、TC明显增加(P<0.05)。葛根芩连汤高剂量组血清HDL水平与模型组相比增加明显(P<0.05)；葛根芩连汤低剂量组与模型组相比,血清LDL水平下降明显(P<0.05)；与模型组相比,葛根芩连汤高、低剂量组血清TC均降低明显(P<0.05)；对于血清甘油三酯,各给药组与模型组之间无统计学意义(P>0.05)。
(4)血清及肝组织IL-6、IL-10、TNF-a水平：与空白组相比,模型组血清及肝组织IL-10显著降低,血清IL-6、TNF-α明显增加(P<0.05)。与模型组相比,葛根芩连汤高、低剂量组可明显增加血清及肝组织IL-10含量,降低血清及肝组织IL-6、TNF-α水平(P
结论
葛根芩连汤高、低剂量均能显著改善高脂饮食饲喂诱导的NASH大鼠肝组织病理,改善脂肪变、炎性因子浸润；能够降低血清转氨酶水平、改善血脂代谢紊乱；能够通过抑制促炎因子、提高抑炎因子两方面动态调控炎症的发展；同时通过对肝组织PPAR γ的调控达到干预大鼠NASH的作用。
3游离脂肪酸诱导NASH体外模型的建立及动态监测
目的
研究游离脂肪酸诱导的非酒精性脂肪性肝炎体外模型,动态观测细胞脂肪变性、炎症、过氧化损伤及糖转运缺陷发生发展的特点。
方法
以HepG2细胞为实验对象,采用不同浓度游离脂肪酸(250μM,500μ M,1000μM)对细胞进行不同时间(12h,18h,24h)的干预刺激,并通过油红0染色,酶联免疫的方法分别对细胞内脂肪变及细胞培养上清液中IL-8, TNF-a, SOD, MDA, GLUT4进行测定评估。
结果
FFA作用的实验各组,随着时间的延长及浓度的增加,细胞脂肪样变、IL-8、TNF-a、SOD、MDA及GLUT4均有不同程度的改变,以24h0.5mM FFA、24h1mM FFA最为明显,与同期空白对照组相比,其在IL-8, TNF-a、SOD、MDA、GLUT4的表达方面均有统计学意义(P<0.05),油红0染色可见典型的“印戒”状。
结论
应用游离脂肪酸诱导建立体外NASH模型成模效果好,重复率高,并提示500μM FFA24h为佳。
4葛根芩连汤对NASH细胞模型的干预研究
目的
探讨葛根芩连汤对非酒精性脂肪性肝炎体外模型的影响。
方法
用500μmol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的葛根芩连汤干预NASH体外模型,MTT法检测葛根芩连汤的安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-a、IL-8及GLUT4。
结果
(1)NASH细胞模型组培养液中TNF-α及IL-8含量明显高于空白对照组(P<0.01),GLUT4含量明显低于空白对照组。
(2)NASH模型组中同时加入葛根芩连汤可明显改善细胞脂肪样变,减少TNF-αIL-8分泌,提高GLUT4含量。
结论
葛根芩连汤能够通过影响TNF-α、IL-8及GLUT4改善NASH体外模型炎症及糖脂代谢。
5葛根素、小檗碱联合用药干预NASH细胞模型的的实验研究
目的
探讨葛根素、小檗碱联合用药干预非酒精性脂肪性肝炎体外模型的相关剂量及作用效果。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的葛根素、小檗碱单药及组合用药干预NASH体外模型。MTT法测定两种单体对非酒精性脂肪性肝病体外模型细胞安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-α、IL-8及GLUT4水平。
结果
(1)500μ mol/LFFA作用NASH模型组,可见细胞胞浆内被染成红色的颗粒状脂滴,并呈“印戒”状形态。不同浓度葛根素、小檗碱单用及联合用药(p500、p1000、p2000、b25、b50、b25p1000、b50p500,单位u mol/L)干预组,可见细胞内红色脂滴有所减少,单体联合用药组优于同浓度单独用药组,基本达到以小剂量联合不次于高剂量单独用药的目的。
(2)500μ mol/L FFA刺激24h后的HepG2细胞,其细胞培养上清液中炎性因子TNF-α及IL-8含量明显增加,GLUT4含量明显下降,与空白组比,差异显著(P<0.05)。
随着小檗碱、葛根素单独使用浓度的增加,IL-8水平较模型组明显降低,以b50、b100、p2000差异明显(P<0.05)；组合用药后b25p1000、b50p500均较模型组有统计学意义(P<0.05),b25p1000较b25效果明显(P<0.05),b50p500较p500、p1000效果明显(P<0.05)。
对于TNF-a,随着小檗碱、葛根素单独使用浓度的增加,其上清液含量显著降低,均较模型组用统计学差异(P<0.05)；组合用药后,TNF-a含量的变化优于单独使用时,b25p1000较p1000、p2000效果明显(P<0.05),b50p500较b50效果明显(P<0.05)。
对于GLUT4,随着小檗碱、葛根素单独使用浓度的增加,其上清液含量显著增加,以b50、b100、p500、p1000、p2000较模型组用统计学差异(P<0.05),且葛根素对于增加GLUT4含量作用优于小檗碱；组合用药后,GLUT4含量的变化优于小檗碱单独使用,b25p1000在加入葛根素后较b25、b50效果明显增强,b50p500较b50、b100效果明显增加(P<0.05)。
结论
葛根素、小檗碱组合用药在改善细胞病理、TNF-α、IL-8、GLUT4水平方面存在协同作用,其组合用药效果较单独用药效果增强。体外实验中,基本能够确定在一定范围内,小檗碱与葛根素组合比例在1：40-1：10为佳。
6葛根素、小檗碱、黄芩苷联合用药干预NASH细胞模型的实验研究
目的
探讨葛根素、小檗碱、黄芩苷联合用药干预非酒精性脂肪性肝炎体外模型的相关剂量及作用效果。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；用不同浓度的黄芩苷单药及葛根素、小檗碱、黄芩苷组合用药干预NASH体外模型。MTT法测定两种单体对非酒精性脂肪性肝病体外模型细胞安全药物浓度范围；油红0染色观察细胞内脂肪变；Elisa法检测细胞培养上清液中TNF-α及IL-8水平。
结果
(1)500μ mol/L FFA作用NASH模型组,可见细胞胞浆内被染成红色的颗粒状脂滴,并呈“印戒”状形态。不同浓度黄芩苷单用及葛根素、小檗碱、黄芩苷联合用药(bai25、bai50、bai100、bai200、b25p1000bai25、b25p1000bai50、b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai,单位100μ mol/L)干预组,可见细胞内红色脂滴有所减少,单体联合用药组效果明显。
(2)500μ mol/L FFA刺激24h后的HepG2细胞,其细胞培养上清液中炎性因子TNF-α及工L-8含量明显增加,与空白组比差异显著(P，0.05)。
随着黄芩苷单独使用浓度的增加,IL-8水平较模型组明显降低,以bai200差异明显(P<0.05)；组合用药后b25p1000bai25、b25p1000bai50、b25p1000bai100、b50p500bai25、 b50p500bai50、b50p500bai100均较模型组有统计学意义(P，0.05),在各比例组合中,以b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai100效果最为明显(P<0.05)。
对于TNF-a,随着黄芩苷单独使用浓度的增加,其上清液含量显著降低,均较模型组用统计学差异(P<0.05);组合用药后,b25p1000bai25、b25pl000bai50、b25p1000bai100、b50p500bai25、b50p500bai50、b50p500bai100均较模型组有统计学意义(P<0.05),且效果优于黄芩苷单独使用时。在各比例组合中,以b50p500bai50、b50p500bai100效果最为明显(P
葛根素、小檗碱、黄芩苷联合用药(b25p1000bai25、b25p1000bai50、b25p1000bai100、 b50p500bai25、b50p500bai50、b50p500bai100)在减少细胞内红色脂滴方面,存在显著协同作用；对于TNF-α、IL-8,通过单体组合使用,可在降低黄芩苷浓度的前提下保证干预效果。体外实验中,基本能够确定在一定范围内,小檗碱、葛根素、黄芩苷组合比例在1：10：1-1：10：2为佳。
7PPAR γ与炎性因子在NASH细胞模型中的相关性研究
目的
明确PPAR y与炎性因子在NASH细胞模型中的关系。
方法
用500μ mol/L的FFA诱导HepG2细胞24h,建立NASH体外模型；造模同时给与不同浓度GW9662／罗格列酮刺激；Elisa法检测细胞培养上清液中TNF-α及IL-8水平,油红0染色测定细胞内脂滴。
结果
(1)与模型组比较,GW966210u M加入后,IL-8及TNF-a含量均增加,但未出现统计学意义。加入罗格列酮2μM后,IL-8及TNF-α含量显著降低,与模型组、gw10组相比,均具有统计学差异(P<0.05)。各单体与gw10组合中,IL-8及TNF-α含量均降低,与模型组、gw10组比较差异显著(P<0.05)。
为了避免因模型带来的非GW9662因素造成的工L-8及TNF-α含量增加,补充了GW9662对未加入FFA诱导的“正常”细胞的作用。结果显示,不同浓度的GW9662均使“正常”细胞上清液中IL-8及TNF-α含量增加,与未加入GW9662组比,差异均具有统计学意义(P<0.05)。
(2)读取OD值进行细胞内脂滴含量分析,GW9662不同浓度较模型组比,脂滴含量均升高,差异显著,具有统计学意义(P<0.05)。
结论
PPAR y阻断剂GW9662可以通过阻断PPAR y促进炎症发展；葛根素、小檗碱、黄芩苷具有潜在的PPAR γ激动剂的作用；PPAR γ与脂质沉积代谢密切相关。
8葛根芩连汤有效组分对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎药效学观察
目的
明确葛根芩连汤有效组分(葛根素、小檗碱、黄芩苷)对高脂饲料饲喂诱导的大鼠非酒精性脂肪性肝炎药效学作用。
方法
以高脂饮食建立大鼠NASH模型,并于造模同时进行药物干预(通过正交表确定组数)；实验第8周末进行血清ALT、AST、TG、HDL-C、LDL-C的测定,同时油红O、HE染色观察肝脏病理切片。
结果
(1)一般情况及病理形态学：模型组大鼠毛色黯淡枯黄,喜团聚蜷卧,饮食量较空白组减少,大便干稀不调,模型组肝组织HE染色存在明显的肝细胞脂肪变性,肝小叶界限不清,肝索结构紊乱,大量空泡及气球样变产生,中性粒细胞浸润,油红0染色可见大量红色脂滴,颜色较深,显示肝组织内脂滴弥漫、含量较多,肝小叶内脂滴融合成片；葛根芩连汤高、低剂量组HE染色病变有不同程度减轻,损伤面积明显减小,胞内炎性细胞浸润减少,空泡及气球样变减少,细胞及小叶结构不同程度恢复,油红0染色脂滴减少。
(2)肝脏酶学：模型组血清ALT、AST均明显高于空白组,有显著差异(P<0.01),各给药组血清ALT、AST水平均较模型组均显著降低(P<0.01),其中F、G、H组在改善血清ALT、AST方面效果明显优于HH组(P<0.01)；D、E组仅在改善AST方面优于HH组(P<0.01)
(3)血脂及肝脂：与空白组相比,模型组HDL显著降低,LDL、TG、TC显著增高,具有统计学差异(P
结论
葛根素为主药的单体组合,对肝组织病理改善,特别是脂滴改善最为明显；以抗炎类小檗碱、黄芩苷为主药的组合,对转氨酶影响最大；不同比例单体组合对血脂的调节都具有积极作用,但随着各单体所占比例的变化,并未见明确的规律性,一些组合的效能基本与葛根芩连汤高剂量组相当,提示组合明确的单体配伍可以在某些方面达到中药原方的效果。
Comprehensive learning of non-alcoholic steatohepatitis (NASH) in both modern medicine and traditional Chinese medicine (TCM) is demonstrated through literature review and experimental study in this paper. Efficacy and mechanism of GeGenQinLian Decoction and its componets are explored in in vivo and in vitro study. The research provided a basic reveal of the mechanism of GeGenQinLian Decoction and its componets, which may give a great contribution to clinical therapeutic levels and new grug discovery.
1Literature review
Through literature analysis in recent10years both at home and abroad, the etiology, pathogenesis, and treatment of modern medicine, also etiology and pathogenesis, syndrome differentiation, physical sciencetreatment progress of TCM have been review.
2Effects and Mechinsm of GeGenQinLian Decoction in NASH Rats induced by High-Fat Diet
Objective
To investigate the efficacy and mechinsm of GeGenQinLian Decoction on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.
Method
The SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by GeGenQinLian Decoction in high and low doses. The change of serum transaminase, serum lipid, serum inflammation factors, liver inflammation factors and the liver pathology had been tested to observe the efficacy. Meanwhile, PPAR γ protein and mRNA in liver were also detected.
Outcome
(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups, together with significantly reduced NAS score. And there was no significant difference between treatment groups.
(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased. The serum ALT and AST decreased both in high and low dose of GeGenQinLian Decoction group.
(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group. The high dose groups of GeGenQinLian Decoction increased the serum HDL-C compared with the model group, while the low dose of GeGenQinLian Decoction decreased the serum LDL-C significantly. Both the high and low dose of GeGenQinLian Decoction decreased serum TC.
(4) Serum and liver IL-6, IL-10and TNF-α:Compared with blank control group, the serum and liver IL-6and TNF-α in model group were obviously increased, while the serum and liver IL-10decrease. GeGenQinLian Decoction can significantly increase the leverl of serum and liver IL-10and decrease the serum and liver IL-6and TNF-α.
(5) liver PPAR γ protein and mRNA expression:Compared with blank control group, the liver PPAR γ protein and mRNA expressions were both declined, which were increased in GeGenQinLian Decoction groups.
Conclusion
GeGenQinLian Decoction can significantly improve liver pathology of the high-fat diet-induced NASH rats; improving steatosis and infiltration of inflammatory cytokines; reducing serum transaminase levels;improving dyslipidemia; and achieving intervention on NASH rats by PPAR γ regulation.
3A Cell Model of Nonalcoholic Steatohepatitis Induced by FFA:Establishment and Dynamic Monitoring
Objective
To establish a cell model of nonalcoholic steatohepatitis (NASH) by free fatty acid (FFA) and to dynamically monitor the pathological features of steatosis, oxidative damage and inflammation in these cells.
Methods
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. For the treatment, palmitic and oleic acid0.1M stock solutions were prepare by dissolving FFA in DMSO (with a final concentration less than0.1%). The cells were exposed to increasing concentrations of a fresh mixture of exogenous FFA (250μmol/L,500μmol/L,1000μmol/L) in molar ratio1:2palmitic:oleic at different time points (12h,18,24h) respectively. Oil red0staining and the IL-8, TNF-α, SOD, MDA, GLUT4 in supernatant were evaluated.
Outcome
Steatosis could be seen in FFA groups to different extents. IL-8, TNF-α, SOD, MDA and GLUT4gradually differed according to the change of stimulation time by different FFA concentrations, especially in the groups with0.5mM and1mM FFA concentrations for24h, of which groups there could be seen a typical steatosis.
Conclusion
A cell model of NASH has been created successfully by FFA.
4Research of Gegenqinlian Decoction on NASH in vitro
Objective
Free fatty acid induced NASH model was used to evaluate the effects of Gegenqinlian Decoction.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of Gegenqinlian Decoction were meanwhile added. MTT, Oil red0staining and the IL-8, TNF-α, GLUT4in supernatant were evaluated.
Outcome
(1) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group (P<0.01).
(2) Gegenqinlian Decoction can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content.
Conclusion
Gegenqinlian Decoction can improve lipid metabolism and inflammation through influencing TNF-a, IL-8and GLUT4.
5Experimental Studies of the Combination Effects of Berberine and Puerarin on NASH in vitro
Objective
To discuss the combination effects of puerarin and berberine on NASH in vi tro.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μ g/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μ mol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin and berberine combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8, TNF-a, GLUT4in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, different concentrations of puerarin and berberine alone and combination (p500, p1000, p2000, b25, b50, b25p1000, b50p500, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.
(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group(P<0.01). Different concentrations of puerarin and berberine combination can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content(P<0.01).
Conclusion
Puerarin and berberine combination can improve cellular pathology, influencing the levels of TNF-a, IL-8and GLUT4, which showed a synergistic effect and better than a single drug effect.
6Experimental Studies of the Combination Effects of Berberine, Puerarin and baicalin on NASH in vitro
Objective
To discuss the combination effects of puerarin, berberine and baicalin on NASH in vitro.
Method
Liver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin, berberine and baicalin combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8and TNF-a in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, different concentrations of puerarin, berberine and baicalin alone and combination (bai25, bai50, bai100, bai200, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.
(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01). Different concentrations of puerarin, berberine and baicalin combination can significantly improve cell degeneration and steatosis and reduce TNF-a, IL-8secretion (P<0.01).
Conclusion
Puerarin, berberine, and baicalin combination can improve cellular pathology, influencing the levels of TNF-a and IL-8, which showed a synergistic effect. The better proportion of berberine, puerarin, baicalin combination was1:10:1-1:10:2in vitro.
7The Related Studies between PPAR γ and Inflammatory Factors on NASH in vitro
Objective
To clarify the relationship between PPAR γ and inflammatory factors.
Method
NASH model in vitro was established using500μmol/L FFA for-24h. The agonists of PPAR γ Rosiglitazone and antaonist of PPAR γ GW9662were meanwhile added.Oil red0staining and the IL-8and TNF-a in supernatant were evaluated.
Outcome
(1) Compared with NASH model group, the GW966210μmol/L group can increase IL-8and TNF-a level; while IL-8and TNF-a were significantly reduced after adding rosiglitazone2μmol/L (P<0.05). In the groups of GW966210μmol/L plus with each monomer, IL-8and TNF-a levels were lowerd compared with the model group and GW966210μmol/L group (P<0.05).
In order to avoid an increase caused by model, GW9662was added to the "normal" cells without FFA. The results showed that different concentrations of GW9662increased IL-8and TNF-a levels compared with the group without GW9662(P<0.05).
(2) The intracellular lipid droplets content was analysized by reading OD values, lipid droplets levels were increased significantly in groups with different concentrations GW9662compared with the model group (P<0.05).
Conclusion
PPAR γ antagonist GW9662can promote inflammation by blocking PPAR γ; puerarin, berberine and baicalin hold the potential effects as PPAR γ agonist; PPAR γ is closely related to lipid metabolism.
8Effects of Components in GeGenQinLian Decoction in NASH Rats Induced by High-Fat Diet
Objective
To investigate the efficacy of components in GeGenQinLian Decoction (puerarin, berberine, baicalin) on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.
Method
The SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by the componts of GeGenQinLian Decoction (number of groups identified by orthogonal table). The change of serum transaminase, serum lipid and the liver pathology had been tested to observe the efficacy.
Outcome
(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups. And there was no significant difference between the all treatment groups.
(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased (P<0.01). The serum ALT and AST decreased both in all treatment groups, in which F, G, H groups were better than HH gtoup in inproving ALT and AST, and D, E group were better than HH group in inproving AST (P<0.01).
(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group (P<0.01). The A, B, D, E, I, HH groups increased the serum HDL-C compared with the model group, in which HH group was better than B, E groups (P<0.01). Compared with the model group, each treatment groups decreased serum LDL levels significantly (P<0.01), in which the HH group was better than E, F, H, I groups (P<0.01). For the serum triglycerides, the level in A, C, D, E, F, G, HH groups decreased compared with model group (P<0.01). For the serum total cholesterol, the level in A, B, C, D, E, F, G, HH group decreased compared with model group (P<0.01).
Conclusion
GeGenQinLian Decoction can significantly improve of the high-fat diet-induced NASH rats and achieve intervention on NASH rats by PPAR γ regulation.
Different proportions of monomer composition gave positive effects to the liver pathology, serum transaminase levels and dyslipidemia. With some certain propotion, it showd an equal effect to the high dose of GeGenQinLian Decoction, suggesting a clear monomer combination could achieve the effect at some aspects with the original formula in traditional Chinese medicine.
引文
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