[This corrects the article DOI: 10.3389/fphar.2021.671152.].

The aqueous decoction, the principal method for preparing Traditional Chinese Medicine formulas (TCMF), is presumed to exert therapeutic effects through the synergy of its constituents. However, the fundamental physicochemical transformations occurring during decoction, which may be crucial for efficacy, are poorly understood. In particular, whether and how endogenous polysaccharides participate in forming pharmacologically active supramolecular assemblies is largely unexplored. This study investigated the classic TCMF Zexie Tang and revealed that thermal decoction drives the self-assembly of an inulin-type fructan from Atractylodis Macrocephalae Rhizoma and triterpenoids (alisol A, alisol B, 23-acetyl-alisol B) from Alismatis Rhizoma into stable, functional supramolecular particles, designated as heated microparticles (HMPs, ∼1 μm). These HMPs markedly enhanced the oral bioavailability of the triterpenoids. Consequently, in a murine model of metabolic dysfunction-associated steatohepatitis (MASH), HMPs exerted a potent effect in alleviating liver injury, steatosis, and dyslipidemia. In vitro reassembly experiments and molecular dynamics simulations indicate that hydrophobic and hydrogen-bonding interactions are key drivers of particle formation. This study demonstrates that endogenous polysaccharides actively participate in supramolecular complexes of Zexie Tang, and that heat-induced fructan-triterpenoid assemblies serve as an important pharmacologically active component that substantially contributes to the therapeutic effects, establishing a materials-based link between traditional decoction and efficacy.

The imbalance between the increasing global prevalence of liver disease and the limited availability of effective drugs highlights the need to develop novel hepatoprotective ingredients. Medicinal fungal polysaccharides (MFPs) have shown promise as hepatoprotective agents owing to their diverse liver-beneficial activities. This review summarizes decade-long advancements in hepatoprotective MFPs studies, including their sources, preparation technologies, chemical structures, hepatoprotective properties and mechanisms, as well as the existing research challenges. A total of 61 hepatoprotective MFPs from 18 species of medicinal fungi have been identified in the past decade. Various advanced techniques for MFPs preparation and characterization are well-established at the laboratory scale. MFPs exhibit hepatoprotective potential through diverse mechanisms in various liver disorders. MFPs offer a promising avenue for the development of scarce hepatoprotective agents, but there are still many challenges to translate the laboratory studies of MFPs into practical applications, including the lack of efficient standard preparation methods for large-scale industrial, lagging standardization and quality control of MFPs, unelucidated systemic biological effects of hepatoprotective MFPs and insufficient market acceptance and awareness of MFPs. Addressing these challenges is expected to facilitate the transition of MFPs from experimental innovation to clinical or commercial application, ultimately benefiting patients with liver disease worldwide.

ETHNOPHARMACOLOGICAL RELEVANCE:Paecilomyces hepiali Chen (Cs-4), recognized as the classic deficiency-tonifiying medicine of traditional Chinese medicine (TCM), is the principal and most valuable medicinal species of Ophiocordyceps sinensis (known as Cordyceps sinensis (Berk.) Sacc., or 'Dong-Chong-Xia-Cao' in Chinese). According to the TCM theories, liver fibrosis results from 'deficiency of healthy qi' and 'accumulation due to yin and deficiency in liver and kidneys', and O. sinensis is traditionally believed to strengthen healthy qi, tonify yin and kidneys. And its efficacy in treating hepatic fibrosis has been widely proven by clinical applications over millennia and contemporary research. Despite extensive research and evidence supporting its beneficial effects on liver fibrosis, the primary active components and underlying mechanism of this substance remain unclear. AIM OF THE STUDY:Extracting Cs-4 polysaccharides using water extraction, alcohol precipitation, and weak anion-exchange chromatography to elucidate structural characteristics, and evaluate efficacy and potential mechanisms in the chronic liver fibrosis mouse models and in vitro studies. MATERIALS AND METHODS:The structural characteristics of Cs-4 polysaccharides were elucidated and mainly characterized by FT-IR spectroscopy and monosaccharide composition. Subsequently, two models on chronic liver fibrosis were set up by intraperitoneal injection of 15 % carbon tetrachloride (CCl4)-olive oil solution, and long-term methionine-choline-deficient (MCD) diet feeding with or without Cs-4 polysaccharides. Then, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic histological evaluations were conducted to assess the effects of Cs-4 polysaccharides. Furthermore, α-smooth muscle actin(α-SMA), collagen type I (Col-Ⅰ), transforming growth factor-β1(TGF-β1), the NLR family, pyrin domain containing protein 3(NLRP3) and Caspase-1 expressions were examined using immunohistochemistry, Real-time quantitative PCR analysis (RT-qPCR) analysis and Western blot to investigate hepatic stellate cells (HSCs) activation and inflammatory responses. Additionally, the human liver stellate cell line-LX2 cells stimulated by TGF-β1 were performed in vitro with varying the presence or absence of CSP-W administration. RESULTS:CSP-W, neutral polysaccharides derived from Cs-4, is composed of Gal, Man, Xyl, Ara, Glc, Fuc, and Rha (molar ratio= 52.4: 35: 1.7: 6.8: 2.8: 0.5: 0.8). Additionally, CSP-W was observed to significantly reduce serum AST, ALT levels and liver histological injury in mice with chronic liver fibrosis induced by CCl4 and an MCD diet. It also decreased collagen fiber deposition and HSCs activation in Sirius Red (SR) or Masson staining, along with a reduction in Col-Ⅰ, α-SMA expressions. Mechanistically, CSP-W lowered TGF-β1 levels, preventing Smad2/3 phosphorylation and activation, and markedly reduced NLRP3, Caspase-1, and IL-1β expressions in both CCl4 and an MCD diet models. Furthermore, CSP-W administration in vitro in TGF-β1-stimulated LX2 cells resulted in the downregulation of markers associated with the TGF-β1/Smad signaling pathway and NLRP3/Caspase-1 signaling pathways. CONCLUSIONS:As neutral polysaccharides, CSP-W exerted a notable curative impact on chronic liver fibrosis in mice induced by CCl4 and MCD diet. The mechanism mainly hindered the activation of HSCs and diminished collagen fibers accumulation through downregulating the TGF-β1/Smad and NLRP3/Caspase-1 signaling pathways in vivo and in vitro. Our findings provided compelling evidence supporting the potential application of CSP-W for managing chronic liver fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE:The ameliorative effect of traditional Chinese medicine (TCM) on hepatic fibrosis has been widely recognized and researched, but studies on the mechanism of action have been hampered by its complex composition, which requires more in-depth studies to elucidate why and how TCM works. The theory of TCM believes that the liver is closely related to blood circulation, and hepatic fibrosis is caused by blood stagnation. Taohong Siwu Decoction (THSW) is a classic formula for nourishing and invigorating blood and has been used clinically for centuries. Current evidence has demonstrated its ameliorative effect on hepatic fibrosis, but the exact mechanism of action remains unclear. AIM OF THE STUDY:Exploring the possible mechanism of the anti-hepatic fibrosis effect of THSW by proteomics and validating with in vivo and in vitro studies. MATERIALS AND METHODS:The carbon tetrachloride (CCl4)-induced fibrosis model was conducted in mice and treated with THSW in vivo with colchicine as the positive control. Then serum biomarker alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathological analysis were evaluated to examine the effects of THSW. And hepatic fibrosis indicators alpha-smooth muscle actin (α-SMA) and Collagen Ⅰ (Col-Ⅰ) were detected by western blotting, immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Additionally, the 4D Label-free quantitative proteomic analysis of liver samples was applied. In vitro, erastin-induced BRL-3A cells, a rat hepatocyte line, were performed as a hepatocyte ferroptosis model and treated with or without drug-containing serum of THSW. Finally, molecular docking was used to verify the binding ability of the main components of THSW to potential targets. RESULTS:THSW treatment significantly ameliorated serum ALT, AST, hydroxyproline (Hyp) content, α-SMA and Col-Ⅰ mRNA expression in fibrosis mice. Further results showed that THSW decreased the malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE) content and increased the glutathione (GSH) content of liver tissue. Notably, proteomic analyses have identified 294 differentially expressed proteins in the THSW-treated group compared to the model group, with 97 proteins up-regulated and 197 down-regulated. Functional analysis of these differential proteins highlights the significant roles of inflammation and oxidative stress. Further validation in vivo and in vitro, THSW significantly improved the protein expression of glutathione S-transferase M1 (GSTM1), down-regulate the expression of transferrin receptor (TFRC), and kelch-like ECH-associated protein 1(Keap1) proteins, and promote the metabolism of GSH. Especially it reduced serum iron levels, increased total iron binding capacity, and up-regulated recombinant solute carrier family 7, member 11 (SLC7A11), nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4) protein expression, suggesting the inhibition of hepatocyte ferroptosis. In addition, the molecular docking results showed that its main components, amygdalin, hydroxysafflor yellow A, paeoniflorin, and albiflorin, possessed good binding ability with Keap1. CONCLUSIONS:THSW represents a novel therapeutic effect on hepatic fibrosis in mice, accompanied by inhibiting hepatocyte ferroptosis. Mechanically, THSW may regulate the glutathione metabolic pathway and TFRC expression through its main ingredients, such as amygdalin, hydroxysafflor yellow A, paeoniflorin, and albiflorin, thereby inhibiting hepatocyte ferroptosis.

BACKGROUND:Bupleuri Radix polysaccharides (BRP) and baicalin (BAI) are key coexisting constituents in the traditional Chinese medicine (TCM) pair Bupleuri Radix-Scutellariae Radix, a classical herbal pair widely used in TCM formulations for treating liver diseases. However, the influence of BRP on BAI's intestinal metabolism, absorption, and therapeutic efficacy remains insufficiently explored. PURPOSE:This study investigated BRP's effects on the oral pharmacokinetics and therapeutic efficacy of BAI in mice with metabolic dysfunction-associated steatohepatitis (MASH) to elucidate underlying mechanisms. METHODS:Mice with MASH, induced by a methionine- and choline-deficient (MCD) diet, received oral BAI with or without BRP. BAI and its metabolite, baicalein (BE), were quantified using LC-MS/MS. 16S rRNA gene sequencing profiled gut microbiota composition, and intestinal β-glucuronidase (β-GUS) activity was measured via ELISA. Bile acid (BA) and polyunsaturated fatty acid (PUFA) profiles were evaluated LC-MS/MS. RESULTS:BRP enhanced BAI's efficacy against liver injury and lipid accumulation. Co-administration elevated serum BAI concentrations and increased BE levels in serum and liver. Intestinal β-GUS activity significantly increased following co-administration. BRP regulated gut microbiota, such as Blautia, RF39, Desulfovibrio, Muribaculaceae and f_Clostridium_methylpentosum_group, which were correlated with β-GUS activity. Moreover, BRP potentiated BAI's regulatory effects on BA and PUFA profiles. CONCLUSION:BRP improved BAI's intestinal absorption and therapeutic efficacy in MASH mice, potentially by modulating gut microbiota and enhancing β-GUS activity, thereby facilitating the deglycosylation of BAI. These findings reveal the potential synergistic mechanism of Bupleuri Radix-Scutellariae Radix pair and propose a feasible approach to improve the bioavailability and therapeutic effect of BAI.

Excessive lipid intake is a major contributor to obesity, and inhibiting intestinal lipid absorption is considered an effective strategy to mitigate this condition. In this study, a pectic polysaccharide designated as RCP-2-3 was obtained from Rubus chingii Hu. unripe fruits and its potential inhibitory effect on intestinal lipid absorption in vivo was investigated. Structural studies revealed that RCP-2-3 (8.54 kDa) is a homogeneous pectic polysaccharide composed of homogalacturonan (HG) and rhamnogalacturonan II (RG-II) domains in a 4.4:4.1 mass ratio. Treatment with RCP-2-3 for 7 days effectively reduced postprandial serum triglyceride (TG) accumulation and lipid absorption in BODIPY-labeled soybean oil-fed mice, accompanied by increased lipid excretion. Specifically, RCP-2-3 increased the excretion of long-chain fatty acids in dietary lipids, such as linoleic acid, oleic acid, palmitic acid, and stearic acid. A 28-day administration of RCP-2-3 reduced weight gain and improved dyslipidemia, and promoted lipid excretion in mice subjected to a high-fat diet and high-fat emulsion. In conclusion, RCP-2-3, a natural pectic polysaccharide consisting of HG and RG-II domains, effectively inhibited intestinal lipid absorption and reduced obesity-related parameters. These findings highlight the potential of RCP-2-3 for application in both the food and pharmaceutical industries.

Ethnopharmacological relevance: Sanghuangporus, the dried fruiting body of Sanghuangporus vaninii (Ljub) L.W. Zhou et Y.C.Dai. As the main species of Sanghuang, it has been well-known and used commonly as a traditional medicinal and edible macrofungi for thousands of years in many countries, including China, Korea and Japan. Although it has good hepatoprotective activity, its potential efficacy and mechanism on liver fibrosis remain elusive. Aim of the study: Total Sanghuangporus vaninii extract (TSH) was prepared by ethanol extraction to investigate its chemical components and to conduct an initial assessment of its efficacy and underlying mechanism in a murine model of liver fibrosis. Materials and methods: The chemical components of TSH were initially analyzed by UHPLC-Q-Orbitrap HRMS. To elucidate the effects of TSH, an in vivo model of fibrosis was established in mice using carbon tetrachloride (CCl4), followed by assessments of serum liver function and histopathological analysis. Besides, indicators related to liver fibrosis, hepatic stellate cells (HSCs) activation, inflammation response and ferroptosis related indicators were detected by western blotting, immunohistochemistry and real-time quantitative PCR (RT-qPCR) analysis. Additionally, the 16S rDNA sequencing and untargeted metabolomics analysis of intestinal microbiota were employed to investigating the role of TSH in gut microbiome. In vitro, the human hepatocyte line L02 was stimulated with erastin and treated with or without TSH to elucidate its underlying mechanism. Results: The administration of TSH significantly improved serum indicators of liver injury in CCl4-induced fibrosis mice, reduced HSCs activation and collagen deposition, while inhibiting the expressions of transforming growth factor-(31(TGF-(31)/Smad signaling pathway. Notably, TSH treatment attenuated hepatocyte ferroptosis and lipid peroxidation both in vivo and in vitro, as evidenced by a marked decrease in liver iron and malondialdehyde (MDA) contents. In particular, TSH was demonstrated to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-glutathione peroxidase 4 (GPX4) signaling pathway, thereby protecting hepatocytes from ferroptosis with a particular enhancement of Nrf2 nuclear transcription. Furthermore, TSH influenced gut microbiota composition and ameliorated intestinal metabolic disorders. The increased abundance of Parasutterella and Olsenellas due to TSH treatment was significantly positively correlated with elevated phosphatidylcholines involved in linoleic acid metabolism, and negatively correlated with the reduction of fatty acyls. And the enrichment of intestinal linoleic acid metabolism presented a negative correlation in liver fibrosis biomarkers.

Hepatocyte ferroptosis promotes the pathogenesis and progression of liver fibrosis. Salvianolic acid B (Sal B) exerts antifibrotic effects. However, the pharmacological mechanism and target has not yet been fully elucidated. In this study, liver fibrosis was induced by CCl4 in wild-type mice and hepatocyte-specific extracellular matrix protein 1 (Ecm1)-deficient mice, which were separately treated with Sal B, ferrostatin-1, sorafenib or cilengitide. Erastin- or CCl4-induced hepatocyte ferroptosis models with or without Ecm1 gene knockdown were evaluated in vitro. Subsequently, the interaction between Ecm1 and xCT and the binding kinetics of Sal B and Ecm1 were determined. We found that Sal B significantly attenuated liver fibrosis in CCl4-induced mice. Ecm1 deletion in hepatocytes abolished the antifibrotic effect of Sal B. Mechanistically, Sal B protected against hepatocyte ferroptosis by upregulating Ecm1. Further research revealed that Ecm1 as a direct target for treating liver fibrosis with Sal B. Interestingly, Ecm1 interacted with xCT to regulate hepatocyte ferroptosis. Hepatocyte ferroptosis in vitro was significantly attenuated by Sal B treatment, which was abrogated after knockdown of Ecm1 in LO2 cells. Therefore, Sal B alleviates liver fibrosis in mice by targeting up-regulation of Ecm1 and inhibiting hepatocyte ferroptosis. The interaction between Ecm1 and xCT regulates hepatocyte ferroptosis.

Total astragalus saponins (TAS) are the main active components of astragali radix, and have potent anti-hepatic fibrosis effect. However, the therapeutic efficacy of TAS and their potential mechanisms in the treatment of primary sclerosing cholangitis (PSC) remain unclear. In this study, two mouse models of PSC, including 3,5-Diethoxycarbonyl-1,4-Dihydro-2,4,6-Collidine (DDC)-induced PSC and Mdr2-/- spontaneous PSC, and the Tgr5-/- mice were used to investigate the therapeutic effect and mechanisms of TAS. Treatment with TAS, particularly with a dose of 56 mg/kg, significantly ameliorated the PSC-related liver injury, cholestasis, collagen deposition, ductular reaction (DR), and fibrosis in the DDC-induced and Mdr2-/-spontaneous PSC mice. Furthermore, treatment with TAS significantly mitigated the PSC-related inflammatory responses in vivo and HIBEpiC cells by inhibiting the expression of TNF-α, IL-6, and IL-1β. Mechanistically, treatment with TAS rescued the PSC-decreased hepatic TGR5 expression to attenuate the NF-κB p65 phosphorylation. Notably, the therapeutic efficacy of TAS on PSC in DDC-induced mice was abrogated in Tgr5-/- mice, suggesting the anti-PSC effect of TAS may depend on enhancing TGR5 expression. In conclusion, TAS ameliorated DR, inflammation and liver fibrosis in both models of PSC mice by rescuing TGR5 expression. Our findings may aid in the design of new therapeutic strategies for the treatment of PSC.

Cordyceps sinensis (Berk.) Sacc. (Ophiocordyceps sinensis) is an edible and medicinal fungus used as a natural superior tonic. It is considered as scarce fungus with a high market demand. Therefore, as an alternative, fermentation technology has been proposed to produce artificial cordyceps (fermented C. sinensis) to address the shortage of cordyceps resources for industrialization and commercial utilization. Numerous studies have proved that polysaccharides are the important bioactive substances in the fermented C. sinensis, but the research data lack systematic review. In this review, current relevant research data regarding the preparation (including extraction, fractionation, and purification), structural characterization (including molecular weight, monosaccharide composition, glycosidic bond type, structural and conformational features), bioactivities, structure-activity relationships (SAR) and applications of polysaccharides from different sources of fermented C. sinensis last decade were analyzed and discussed. The findings highlight that the most commonly employed methods for preparing fermented Cordyceps sinensis polysaccharides (FCSPs) involve water extraction and alcohol precipitation, combing with sophisticated chromatographic techniques such as ion exchange and gel permeation chromatography. From these processes, 34 different polysaccharides were identified including 5 glucans and 7 heteropolysaccharides that were thoroughly characterized. FCSPs exhibited a broad spectrum of biological activities, ranging from antioxidant and renal protective effects to immunomodulatory, antitumor, and hypolipidemic properties. The structure-activity relationships (SAR) demonstrated that key factors, such as molecular weight, monosaccharide composition and glucosidic bond types, play critical roles in determining the bioactivity of FCSPs. Nevertheless, there remain unknown elements that continue to influence SAR, leaving room for further exploration. Furthermore, the limitation of existing studies and some new perspectives for future investigations on FCSPs were proposed.

Background: The activation of HIF-1α/CXCR4 pathway in liver sinusoidal endothelial cells (LSECs) could downregulate CXCR7, leading to the capillarization of LSECs to promote hepatic fibrosis. However, the mechanism between CXCR4 and CXCR7 is still undefined. The aim is to investigate the role of PDGF-BB in the dedifferentiation of LSECs and hepatic stellate cells (HSCs) activation. Methods: The activation of HIF-1α/CXCR4 pathway in two kinds of liver fibrosis models were observed. The effects of HIF-1α, CXCR4, PDGF-BB on the dedifferentiation of LSECs were investigated by using the inhibitors of HIF-1α, CXCR4 or PDGFR-β separately or transfecting with a CXCR4 knockdown lentiviral vector. In addition, the relationship between LSECs and HSCs was demonstrated by co-culture of LSECs and HSCs using the transwell chamber. Results: CXCR4 upregulation and CXCR7 downregulation were accompanied by LSECs capillarization and HSCs activation both in CCl4-induced and BDL-induced fibrotic liver. In vitro, downregulation of HIF-1α significantly descreased CXCR4 and CD31 expression, and enhanced the expressions of CXCR7, CD44 and LYVE1. Downregulation of CXCR4 in LSECs significantly downregulated PDGF-BB, PDGFR-β and CD31, and enhanced CXCR7, CD44 and LYVE1 expression, while the expression of HIF-1α did not change significantly. STI571, a PDGF receptor inhibitor, could significantly downregulate PDGFR-β and increase the expression of CXCR7 to inhibit the dedifferentiation of LSECs. In addition, alleviateion the dedifferentiation of LSECs could decrease the expression of PDGFR-β of HSCs, then inhibiting the activation of HSCs. Conclusions: This study revealed that HIF-1α/CXCR4/PDGF-BB/CXCR7 axis promoted the dedifferentiation of LSECs, consequently triggering HSCs activation and liver fibrosis.

目的 探究黄芪总苷对胆汁淤积性肝纤维化小鼠的影响及其作用机制.方法 采用 2 种胆汁淤积性肝纤维化模型,0.1%DDC喂养C57BL/6 小鼠 8 周,第 5 周起灌胃给予黄芪总苷 112、56 mg/kg和奥贝胆酸 4 周;Mdr2-/-小鼠 8周龄时灌胃给予黄芪总苷 56、28 mg/kg和奥贝胆酸 3 周,对照组和模型组分别灌胃给予等体积蒸馏水.给药结束后检测各组小鼠肝质量与肝脏系数、血清肝功能、肝组织病理学变化和肝组织纤维化相关指标的表达.结果 与对照组比较,DDC和Mdr2-/-模型组小鼠肝质量和肝脏系数均升高(P＜0.01),血清ALT、AST、ALP活性和TBA、胆红素水平均升高(P＜0.01),肝组织病理可见结构紊乱、大量炎性细胞浸润,汇管区胶原沉积,胶原阳性面积和肝组织HYP水平均增加(P＜0.01),肝组织 α-SMA、Col1a1、Col4a1 和 TGF-β1 表达均升高(P＜0.01),SIRT1 表达降低(P＜0.01).在 2 种模型中,与模型组比较,黄芪总苷 56 mg/kg组小鼠肝质量和肝脏系数均降低(P＜0.05,P＜0.01),血清ALT、AST、ALP活性和TBA、胆红素等水平均降低(P＜0.05,P＜0.01),肝脏损伤明显改善,胶原沉积减少,胶原阳性面积和肝组织HYP水平均减少(P＜0.05,P＜0.01),肝组织α-SMA、Col1a1、Col4a1 和TGF-β1 表达降低(P＜0.05,P＜0.01),SIRT1 表达升高(P＜0.05,P＜0.01).结论 56 mg/kg黄芪总苷可有效改善胆汁淤积性肝纤维化,其作用机制与促进SIRT1 表达相关.

目的：观察不同剂量野百合碱（MCT）诱导大鼠肝窦阻塞综合征（HSOS）的病理变化，探讨造模剂量-时间致肝损伤的病理特征，并解析其部分作用机制。方法：将72只雄性SD大鼠随机分为正常组（n=12）,MCT低、中、高剂量组（每组n=20）。MCT低、中、高剂量组分别采用MCT 80、120、160 mg·kg-1灌胃一次制备模型，各剂量组大鼠于造模48、120 h处死取材。观测各组大鼠的存活率，检测大鼠体质量、肝质量与血清肝功能变化，采用扫描电镜、苏木素-伊红（HE）染色及天狼星红（SR）染色观察肝组织病理变化，微板法检测肝组织匀浆谷胱甘肽S转移酶（GST）、总超氧化物歧化酶（T-SOD）活性、丙二醛（MDA）含量，实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）和免疫组化检测肝组织相关指标的表达。结果：与正常组比较，MCT各组血清丙氨酸氨基转移酶/天冬氨酸氨基转移酶（ALT/AST）活性随造模剂量增加而升高（P＜0.05,P＜0.01）；随造模时间增加，MCT低剂量组血清ALT和AST活性均下降（P＜0.01）,MCT中、高剂量组ALT和AST活性均显著升高（P＜0.01）。HE染色显示MCT低、中、高剂量组肝组织肝细胞坏死、炎性细胞浸润及红细胞淤积，电镜下可见肝窦内皮窗孔扩大，“筛状”结构消失，其损伤程度随剂量提高而加重。与正常组比较，MCT低、中、高剂量组肝窦内皮细胞标志物CD44表达减少（P＜0.05,P＜0.01）。SR染色显示造模48 h MCT各组未见阳性染色，120 h MCT各组可见汇管区及肝窦胶原沉积。与正常组比较，MCT各剂量组肝组织MDA含量及GST活性增加，T-SOD活性降低，以MCT中、高剂量组变化显著（P＜0.01）；且造模120 h MCT各组变化呈现剂量依赖性（P＜0.01）。免疫组化及Western blot结果显示，与正常组比较，促炎巨噬细胞标志物CD68蛋白表达随剂量升高而增加（P＜0.01），抗炎巨噬细胞CD163蛋白及mRNA表达随剂量升高而减少（P＜0.01）。与正常组比较，造模后MCT中、高剂量组磷酸化核转录因子-κB/核转录因子-κB（p-NF-κB/NF-κB）、磷酸化蛋白激酶B/蛋白激酶B（p-Akt/Akt）明显升高（P＜0.05,P＜0.01）。MCT造模后肝组织α-平滑肌肌动蛋白（α-SMA）蛋白表达随时间延长、剂量升高增加，α-SMA、Ⅰ型胶原蛋白基因α1（Col 1a1）和Ⅳ型胶原蛋白基因α1（Col 4a1）mRNA表达也随时间延长、剂量升高而明显增加（P＜0.05,P＜0.01）。TUNEL染色结果显示造模后凋亡细胞数量随造模剂量升高而增加，B细胞淋巴瘤-2（Bcl-2）蛋白与Bcl-2相关X蛋白（Bax）比值显著降低（P＜0.01）。结论：不同剂量MCT灌胃诱导的大鼠肝窦阻塞综合征，病变随剂量增加而加重。80 mg·kg-1MCT导致的肝脏损伤可自愈，超过120 mg·kg-1的MCT导致的肝脏损伤会随着时间的延长而加重，甚至出现纤维化、死亡。MCT导致大鼠HSOS的病理机制可能是MCT引发肝组织内强烈的氧化应激，激活促炎巨噬细胞大量分泌炎症因子，进而激活NF-κB/Akt信号通路，导致严重的细胞损伤和死亡。

目的:观察不同剂量野百合碱(MCT)诱导大鼠肝窦阻塞综合征(HSOS)的病理变化,探讨造模剂量-时间致肝损伤的病理特征,并解析其部分作用机制.方法:将72只雄性SD大鼠随机分为正常组(n=12),MCT低、中、高剂量组(每组n=20).MCT低、中、高剂量组分别采用MCT 80、120、160 mg·kg-1灌胃一次制备模型,各剂量组大鼠于造模48、120 h处死取材.观测各组大鼠的存活率,检测大鼠体质量、肝质量与血清肝功能变化,采用扫描电镜、苏木素-伊红(HE)染色及天狼星红(SR)染色观察肝组织病理变化,微板法检测肝组织匀浆谷胱甘肽S转移酶(GST)、总超氧化物歧化酶(T-SOD)活性、丙二醛(MDA)含量,实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)和免疫组化检测肝组织相关指标的表达.结果:与正常组比较,MCT各组血清丙氨酸氨基转移酶/天冬氨酸氨基转移酶(ALT/AST)活性随造模剂量增加而升高(P＜0.05,P＜0.01);随造模时间增加,MCT低剂量组血清ALT和AST活性均下降(P＜0.01),MCT中、高剂量组ALT和AST活性均显著升高(P＜0.01).HE染色显示MCT低、中、高剂量组肝组织肝细胞坏死、炎性细胞浸润及红细胞淤积,电镜下可见肝窦内皮窗孔扩大,"筛状"结构消失,其损伤程度随剂量提高而加重.与正常组比较,MCT低、中、高剂量组肝窦内皮细胞标志物CD44表达减少(P＜0.05,P＜0.01).SR染色显示造模48 hMCT各组未见阳性染色,120 hMCT各组可见汇管区及肝窦胶原沉积.与正常组比较,MCT各剂量组肝组织MDA含量及GST活性增加,T-SOD活性降低,以MCT中、高剂量组变化显著(P＜0.01);且造模120 hMCT各组变化呈现剂量依赖性(P＜0.01).免疫组化及Western blot结果显示,与正常组比较,促炎巨噬细胞标志物CD68蛋白表达随剂量升高而增加(P＜0.01),抗炎巨噬细胞CD163蛋白及mRNA表达随剂量升高而减少(P＜0.01).与正常组比较,造模后MCT中、高剂量组磷酸化核转录因子-κB/核转录因子-κB(p-NF-κB/NF-κB)、磷酸化蛋白激酶B/蛋白激酶B(p-Akt/Akt)明显升高(P＜0.05,P＜0.01).MCT造模后肝组织α-平滑肌肌动蛋白(α-SMA)蛋白表达随时间延长、剂量升高增加,α-SMA、Ⅰ型胶原蛋白基因α1(Col 1a1)和Ⅳ型胶原蛋白基因α1(Col 4a1)mRNA表达也随时间延长、剂量升高而明显增加(P＜0.05,P＜0.01).TUNEL染色结果显示造模后凋亡细胞数量随造模剂量升高而增加,B细胞淋巴瘤-2(Bcl-2)蛋白与Bcl-2相关X蛋白(Bax)比值显著降低(P＜0.01).结论:不同剂量MCT灌胃诱导的大鼠肝窦阻塞综合征,病变随剂量增加而加重.80 mg·kg-1 MCT导致的肝脏损伤可自愈,超过120 mg·kg-1 的MCT导致的肝脏损伤会随着时间的延长而加重,甚至出现纤维化、死亡.MCT导致大鼠HSOS的病理机制可能是MCT引发肝组织内强烈的氧化应激,激活促炎巨噬细胞大量分泌炎症因子,进而激活NF-κB/Akt信号通路,导致严重的细胞损伤和死亡.

OBJECTIVE:To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.METHODS:Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.RESULTS:High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).CONCLUSIONS:Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

目的 基于均匀设计法探索扶正化瘀方(Fuzheng Huayu,FZHY)有效成分组合及配伍效应.方法 以扶正化瘀方提取物中含量最高的丹参化合物成分F-01及主要入血桃仁活性成分F-02及五味子成分F-03为研究对象,运用均匀设计法,以肝组织羟脯氨酸(hydroxyproline,Hyp)含量和天狼猩红(Sirius red,SR)染色胶原半定量为筛选指标,采用四氯化碳(carbon tetrachloride,CCl4)和胆管结扎(bile duct ligation,BDL)诱导大鼠肝纤维化模型,经回归分析获得"最佳配方",并进行再验证.结果 ①均匀设计方案中的JY5组合可显著降低CCl4和BDL诱导的肝纤维化大鼠血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬酸氨基转移酶(aspartate aminotransferase,AST)活性,提高血清白蛋白(albumin,ALB)含量,降低肝组织羟脯氨酸(hydroxyproline,Hyp)含量及胶原面积半定量;其回归方程则提示配方P(F-01(32 mg·kg-1)+F-02(0.5 mg·kg-1)+F-03(0.5 mg·kg-1))是三者降低CCl4肝纤维化大鼠肝组织Hyp含量和BDL肝纤维化大鼠肝组织胶原沉积从而发挥抗肝纤维化作用的最佳配比.②验证实验表明配方P对CCl4和BDL肝纤维化大鼠血清ALT、AST、ALB及肝组织Hyp含量及胶原面积半定量并无显著改善作用,而JY5的抗肝纤维化作用在CCl4和BDL肝纤维化大鼠实验中均得到再证实.结论 ①F-01(16 mg·kg-1)+F-02(0.5 mg·kg-1)+F-03(2 mg·kg-1)组合可显著改善CCl4和BDL大鼠肝纤维化,疗效与FZHY原方相当.②均匀设计对量效关系呈非线性的中药复方效应成分的研究可能有其一定局限性.

Advanced liver fibrosis can lead to cirrhosis, resulting in an accelerated risk of hepatocellular carcinoma and liver failure. Fuzheng Huayu formula (FZHY) is a traditional Chinese medicine formula treated liver fibrosis in China approved by a Chinese State Food and Drug Administration (NO: Z20050546), composed of Salvia Miltiorrhiza bge., Prunus davidiana (Carr.) Franch., cultured Cordyceps sinensis (BerK.) Sacc. Mycelia, Schisandra chinensis (Turcz.) Baill., Pinus massoniana Lamb., and Gynostemma pentaphyllum (Thunb.) Makino. However, the main active substances and mechanism of FZHY are unclear. The aim of this study is to identify a novel anti-fibrotic compound, which consists of the main active ingredients of FZHY, and investigate its mechanism of pharmacological action. The main active ingredients of FZHY were investigated by quantitative analysis of FZHY extracts and FZHY-treated plasma and liver in rats. The anti-fibrotic composition of the main active ingredients was studied through uniform design in vivo , and its mechanism was evaluated in carbon tetrachloride (CCl 4 )- and bile duct ligation (BDL)-induced liver fibrosis models in rats and mice, and transforming growth factor beta 1-induced LX-2 cell activation model in vitro . A novel Chinese medicine, namely JY5 formula, consisting of salvianolic acid B, schisantherin A, and amygdalin, the main active ingredients of FZHY, significantly alleviated hepatic hydroxyproline content and collagen deposition in CCl 4 -and BDL-induced fibrotic liver in rats and mice. In addition, JY5 inhibited the activation of hepatic stellate cells (HSCs) by inactivating Notch signaling in vitro and in vivo . In this study, we found a novel JY5 formula, which exerted anti-hepatic fibrotic effects by inhibiting the Notch signaling pathway, consequently suppressing HSCs activation. These results provide an adequate scientific basis for clinical research and application of the JY5 formula, which may be a potential novel therapeutic candidate for liver fibrosis.