Jiawei Taohe Chengqi Decoction attenuates hepatic fibrosis by preventing activation of HSCs through regulating Src/ERK/Smad3 signal pathway.
Jiawei Taohe Chengqi Decoction attenuates hepatic fibrosis by preventing activation of HSCs through regulating Src/ERK/Smad3 signal pathway.
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DOI：
10.1016/j.jep.2022.116059
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发表时间：
2022-12
影响因子：
5.4
通讯作者：
Yan-Ning Huang;Zhi-li Wang;Yi‐Ran He;Linmao Ye;Wen-Qin Guo;Jun-jie Zhang
中科院分区：
文献类型：
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作者：
Yan-Ning Huang;Zhi-li Wang;Yi‐Ran He;Linmao Ye;Wen-Qin Guo;Jun-jie Zhang
加味桃核承气汤（JTCD）是由中医经典文献《伤寒论》中桃核承气汤加减而成的中药方剂。临床和药理研究表明，JTCD对肝性脑病、非酒精性脂肪肝、肝硬化腹水有治疗作用，并能减轻大鼠急性肝损伤。本课题组前期的研究证实，中药复方具有减轻肝纤维化和激活肝星状细胞的作用。本研究旨在探讨Src信号对肝纤维化和HSCs活化的作用机制，以及JTCD是否通过影响Src信号抑制肝纤维化和HSCs活化。对照组、模型组、SARA组、JTCD低剂量组、JTCD中剂量组、JTCD高剂量组。建立四氯化碳（CCL 4）诱导的小鼠肝纤维化模型，JTCD组灌胃给予相应剂量的JTCD，SARA组给予Saracatinib，对照组给予生理盐水，每天1次，连续4周。采用UPLC-Q-TOF-MS联用技术分析了JTCD的化学成分。采用HE染色、Masson染色和天狼星红染色观察肝纤维化的特点。全自动生化分析仪检测血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）水平。Western-blot和免疫组化染色（IHC）检测蛋白表达。在体外实验中，我们利用shRNA技术敲低永生化人肝星状细胞系LX-2中Src的表达，然后用ERK 1/2激动剂/抑制剂和转化生长因子β1（TGF-β1）处理后的含JTCD血清进行干预。免疫荧光和western-blot检测蛋白表达。通过伤口愈合试验（wound-healingassay. ResultsWe）对HSC的迁移特性进行了评估。结果JTCD水提取物中鉴定出135种化学成分，JTCD水提取物中含有多种抗肝纤维化成分。与模型组相比，JTCD组和SARA组肝纤维化指标明显改善，血清ALT和AST水平明显降低，免疫组化染色和western blot结果显示JTCD可降低α-平滑肌肌动蛋白（α-SMA）、磷酸化Src（Tyr 416）、磷酸化ERK 1/2和磷酸化Smad 3的表达。Western blot和免疫荧光检测结果显示，JTCD含药血清能显著降低α-SMA、磷酸化Src（Tyr 416）、磷酸化ERK 1/2和磷酸化Smad 3的蛋白表达，并抑制LX-2的迁移和活化。此外，经Src-shRNA、ERK 1/2激动剂/抑制剂和JTCD含药血清干预后，Western-blot结果显示Src/ERK/Smad 3信号在肝纤维化和HSC中起重要作用，结论Src激酶通过调节ERK/Smad 3信号通路促进肝纤维化和HSCs活化，JTCD可能通过抑制Src/ERK/Smad 3信号通路抑制HSCs活化而减轻肝纤维化。Smad 3信号通路更重要的是，JTCD减轻肝纤维化和HSC活化的机制是通过抑制Src/ERK/Smad 3信号通路。
Ethnopharmacological relevanceJiawei Taohe Chengqi Decoction (JTCD) is a Traditional Chinese Medicine (TCM) formula modified from Taohe Chengqi Decoction in the classic ancient literature of TCM “Treatise on Febrile Diseases”. Clinical and pharmacological studies have shown that JTCD has a therapeutic effect on hepatic encephalopathy, non-alcoholic fatty liver, cirrhotic ascites, and can alleviate acute liver injury in rats. Our previous studies confirmed that JTCD could alleviate hepatic fibrosis and activation of hepatic stellate cells (HSCs). However, its mechanism remains unclear.Aim of the studyThis study aimed to elucidate the mechanism of Src Signal on hepatic fibrosis and HSCs activation, and whether JTCD inhibited hepatic fibrosis and HSCs activation through affecting Src Signal.Materials and methodsIn vivo, sixty specific pathogen free male C57/BL6 mice were divided into following six groups: Control group, Model group, SARA group, JTCD low dose group, JTCD medium dose group and JTCD high dose group. Then we established a carbon tetrachloride (CCL4)-induced hepatic fibrosis mice model, each JTCD group was given the corresponding dose of JTCD by gavage, the SARA group was given Saracatinib and the control group was given saline, once a day for 4 consecutive weeks. UPLC-Q-TOF-MS analyzed chemical components of JTCD. Pathological examination including Hematoxylin and Eosin (H&E), Masson and Sirius red staining was used to observe the characteristic of hepatic fibrosis. Automatic biochemical analyzer detected the levels of alanine aminotransfease (ALT), and aspartate transaminase (AST) in serum. Western-blot and immunohistochemical staining (IHC) detected protein expression. In vitro, we used shRNA to knock down the expression of Src in immortalized human hepatic stellate cell line (LX-2), then intervened with ERK1/2 agonists/inhibitors and JTCD-containing serum after transforming growth factor β1 (TGF-β1) treatment. Immunofluorescence and western-blot detected protein expression. The migratory characteristic of HSCs was assessed by wound-healing assay.ResultsWe identified 135 chemical components in the water extract of JTCD, and the water extract of JTCD contains a variety of anti-hepatic fibrosis components. Compared to the model group, hepatic fibrosis performance was significantly improved, the serum levels of ALT and AST were significantly decreased in JTCD groups and SARA group, IHC staining and western blot results indicated that JTCD decreased the expressions of α-smooth muscle actin (α-SMA), phospho-Src (Tyr416), phospho-ERK1/2 and phospho-Smad3. In vitro, JTCD-containing serum could significantly decrease the protein expressions of α-SMA, phospho-Src (Tyr416), phospho-ERK1/2 and phospho-Smad3 according to the results of western-blot and immunofluorescence, in addition, JTCD-containing serum inhibited the mobility and activation of LX-2. What's more, after intervening with Src-shRNA, ERK1/2 agonists/inhibitors and JTCD-containing serum, the western-blot results showed that Src/ERK/Smad3 signal has an important role in hepatic fibrosis and HSCs, and JTCD attenuates hepatic fibrosis by preventing activation of HSCs through regulating Src/ERK/Smad3 signal pathway.ConclusionsThe results showed that Src kinase promoted hepatic fibrosis and HSCs activation through the ERK/Smad3 signal pathway. More importantly, the mechanism by which JTCD attenuated hepatic fibrosis and HSCs activation was by inhibiting the Src/ERK/Smad3 signal pathway.