Fuzheng Huayu Recipe Research Articles

Fuzheng Huayu recipe inhibits bleomycin-induced pulmonary fibrosis in rats by inhibiting M2 polarization of macrophages via the oxidative phosphorylation pathway

Fuzheng Huayu recipe (FZHYR) is a Chinese patent medicine for the treatment of fibrosis. The effects of FZHYR on pulmonary fibrosis and macrophage polarization were investigated in vitro. FZHYR inhibited pulmonary inflammation and fibrosis and M2 polarization of macrophages in bleomycin-induced pulmonary fibrosis (BPF) of rat model. Differentially expressed genes were screened by high-throughput mRNA sequencing and GSEA showed that oxidative phosphorylation (OXPHOS) was correlated with BPF. FZHYR inhibited expressions of Ndufa2 and Ndufa6 in lung tissues of BPF rats. These findings suggest that OXPHOS pathway serves as a possible target for pulmonary fibrosis therapy by FZHYR.

Fuzheng Huayu recipe alleviates liver fibrosis via inhibiting NLRP3 inflammasome activation in macrophages

Ethnopharmacological relevanceFuzheng Huayu recipe (FZHY) is a commonly used Traditional Chinese Medicine formula for treating liver fibrosis in clinical settings. Despite its widespread use, the specific curative effects and underlying pharmacological mechanisms of FZHY in treating liver fibrosis are not yet fully understood. Aim and studyThis study aims to investigate the antifibrotic mechanism of FZHY treatment by exploring its effects on the activation of NOD-like receptor protein 3 (NLRP3) inflammasome in macrophages. Materials and methodsIn order to investigate the impact of FZHY on the activation and priming of NLRP3 inflammasome in clinical trials and animal experiments using immunohistochemistry and Western blotting. Twenty-four C57BL/6 mice were used to induce liver fibrosis by feeding a diet that contained 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). To study inflammasome function, Lipopolysaccharide (LPS)/adenine triphosphate (ATP) induced NLRP3 inflammasome activation was induced in bone marrow-derived macrophages (BMDMs) isolated from wild mice. The effects of macrophage NLRP3 inflammasome activation on the function of hepatic stellate cells (HSCs) were explored by treating primary HSCs with preconditioned media from BMDMs culture. ResultsFZHY treatment resulted in the downregulation of NLRP3 protein expression and inhibition of its priming and activation in both human fibrotic livers and DDC-induced liver fibrosis. Furthermore, FZHY was observed to block the activation of the NLRP3 inflammasome pathway, which can lead to excessive inflammatory cytokine release in supernatants and cell lysates in response to LPS and ATP. Lastly, treatment with FZHY was able to inhibit the activation of HSCs induced by supernatants from macrophages. ConclusionsFZHY has been shown to potentially prevent NLRP3 inflammasome activation in macrophages which can result in the suppression of HSCs activation. Ultimately, these effects may lead to the improvement of liver fibrosis. The ability of FZHY to act on this novel mechanism represents an important aspect of its therapeutic potential for liver fibrosis.

A herbal product inhibits carbon tetrachloride-induced liver fibrosis by suppressing the epidermal growth factor receptor signaling pathway.

Fuzheng Huayu formula (FZHY), composed of Salvia miltiorrhiza Bunge, Cordyceps sinensis, the seed of Prunus persica (L.) Batsch, the pollen of Pinus massoniana Lamb, Gynostemma pentaphyllum (Thunb.) Makino and the fruit of Schisandra chinensis (Turcz.) Baill, is a Chinese herbal compound with demonstrated clinical benefits in liver fibrosis (LF). However, its potential mechanism and molecular targets remain to be elucidated. This study was designed to evaluate the anti-fibrotic role of FZHY in hepatic fibrosis and to elucidate the potential mechanisms. Network pharmacology was assayed to identify the interrelationships among compounds of FZHY, potential targets and putative pathways on anti-LF. Then the core pharmaceutical target for FZHY against LF was verified by serum proteomic analysis. Further in vivo and in vitro assays were performed to verify the prediction of the pharmaceutical network. The network pharmacology analysis revealed that a total of 175 FZHY-LF crossover proteins were filtered into a protein-protein interaction (PPI) network complex and designated as the potential targets of FZHY against LF, and the Epidermal Growth Factor Receptor (EGFR) signaling pathway was further explored according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then analytical studies were validated by carbon tetrachloride (CCl4)-induced model in vivo. We found FZHY could attenuate CCl4-induced LF, especially decrease p-EGFR expression in α-Smooth Muscle Actin (α-SMA)-positive hepatic stellate cell (HSC) and inhibit the downstream of the EGFR signaling pathway, especially Extracellular Regulated Protein Kinases (ERK) signaling pathway in liver tissue. We further demonstrate that FZHY could inhibit Epidermal Growth Factor (EGF)-induced HSC activation, as well as the expression of p-EGFR and the key protein of the ERK signaling pathway. FZHY has a good effect against CCl4-induced LF. The action mechanism was associated with the down-regulation of the EGFR signaling pathway in activated HSCs.

Cyp4a12-mediated retinol metabolism in stellate cells is the antihepatic fibrosis mechanism of the Chinese medicine Fuzheng Huayu recipe

BackgroundHepatic stellate cells (HSCs), which contain multiple retinol-containing lipid droplets, are important profibrotic cells in liver fibrosis. Under Cyp4a12a/b oxidation, HSC activation was accompanied by the downregulation of genes involved in retinol metabolism, inducing RAE-1 production. By eliminating activated HSCs, NK cells expressing the activating receptor NKG2D are recruited to alleviate fibrosis. FZHY was found to significantly reduce the severity of liver fibrosis by inhibiting the activation and proliferation of HSCs. The molecular processes that govern retinol metabolism, on the other hand, are largely unexplored. This study focused on the regulation of Cyp4a12a/b by FZHY to elucidate the antifibrotic molecular mechanisms underlying the effect of FZHY on retinol metabolism.MethodsTo investigate mechanisms and altered pathways of FZHY against carbon tetrachloride (CCl4)-induced liver fibrosis based on transcriptomics data. Bioinformatics analysis was used to screen its pharmacological targets. The predicted targets were confirmed by a series of in vitro and in vivo experiments, including mass spectrometry, in situ hybridization, immunofluorescence assays and real-time PCR. Then, the results were further characterized by recombinant adenovirus vectors that were constructed and transfected into the cultured primary HSCs.ResultsTranscriptomics revealed that Cyp4a12a/b is nearly completely lost in liver fibrosis, and these effects might be partially reversed by FZHY therapy recovery. In vitro and in vivo studies indicated that Cyp4a12a/b deletion disrupted retinol metabolism and lowered Rae-1 expression. Activated HSCs successfully escape recognition and elimination by natural killer (NK) cells as a result of reduced Rae-1. Notablely, we discovered that FZHY may restore the Cyp4a12a/b capability, allowing the recovery of the cytotoxic function of NK cells against HSCs, and thereby reducing hepatic fibrosis by suppressing HSC activation.ConclusionOur findings revealed a new role for Cyp4a in retinol metabolism in the development of hepatic fibrosis, and they highlight Cyp4a12/Rae-1 signals as possible therapeutic targets for antifibrotic medicines.

Pharmacological mechanisms of Fuzheng Huayu formula for Aristolochic acid I-induced kidney fibrosis through network pharmacology.

Renal fibrosis, characterized by the destruction of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins, is a common outcome of chronic renal diseases and has a wide spectrum of etiologies. Fibrosis can affect any organ and has similar pathological mechanisms. Fuzheng Huayu formula (FZHY), as the approved anti-liver fibrosis medicine in China, also can inhibit the kidney fibrosis induced by HgCl2 or unilateral ureteral obstruction. However, little is known about the mechanisms underlying the beneficial effects of FZHY on renal fibrosis. This study aimed to identify the mechanisms of FZHY acts on renal fibrosis through network pharmacological analysis and in vivo experiments. Data from online databases were mined and screened to predict the target related genes of FZHY acts on renal fibrosis. The STRING and Cytoscape were used to construct the protein-protein interaction (PPI) networks for FZHY and CKD target proteins. Mouse models with CKD induced by Aristolochic Acid I (AAI) were used to validate the effects of FZHY on renal fibrosis and their underlying mechanisms by detecting kidney function, renal fibrosis, and related intersection genes. A total of 129 FZHY-CKD crossover proteins were filtered and constructed into a protein-protein interaction network complex and designated as the potential targets of FZHY. One of the highest-scoring genes, FOS, and its related signaling pathways were more activated in CKD. The results demonstrated that FZHY can exert an anti-renal fibrosis effect by improving the levels of serum creatinine and blood urea nitrogen and alleviating excessive collagen deposition in kidney tissue, FZHY also could reduce the levels of TNF-α, IL-1β, and IL-6 and inhibit the expression of MAPK/FOS signal molecules. Our study findings provide insights into predicting the effects of FZHY on CKD through network pharmacology. FZHY can protect the kidney from inflammatory injury caused by AAI and can antagonize inflammatory factor-stimulated MAPK/FOS activation in fibrotic kidneys. These effects constitute the mechanisms of FZHY for renal fibrosis.

Fuzheng Huayu Recipe and its active compounds inhibited HBeAg production by promoting TOMM34 gene expression in HBV-infected hepatocytes.

Background and aim: Fuzheng Huayu Recipe (FZHY) is a Chinese patent medicine (approval No. Z20020074) included in the national medical insurance catalogue, which is mainly used for anti-hepatic fibrosis treatment of hepatitis B virus (HBV) induced liver fibrosis and liver cirrhosis. In clinical practice, we discovered that FZHY might also have a direct anti-HBV effect on inhibiting HBeAg production, but the mechanism underlying was unclear. This study aimed to clarify the molecular mechanism of the inhibition effect of FZHY on HBeAg production. Methods: The decrease degree of serum HBeAg titer in FZHY + entecavir (ETV) group patients were analyzed through clinical data. C57BL/6N-Tg (1.28HBV)/Vst HBV transgenic mice were used for in vivo experiments. HepG2. 2.15 cells (wild-type HBV replication cells) were used for in vitro experiments. Results: The clinical study results showed that the decrease degree of serum HBeAg titer in FZHY+ETV group was significantly higher than that in ETV group after 48 weeks treatment. In vivo experiments results showed that FZHY could significantly reduce the serum HBeAg titer in HBV transgenic mice, and promote HBeAg seroconversion. In vitro experiments results showed that FZHY could reduce HBeAg titer dependently, but it did not significantly inhibit the expression of HBsAg and HBV-DNA. Further cell experiments in vitro discovered that TOMM34 might be the key target for FZHY to inhibit HBeAg production. The subsequent pharmacological screening experiment of 20 active compounds in FZHY showed that quercetin, baicalin and cordycepin could promote the expression of TOMM34 gene and reduce the production of HBeAg. Conclusion: In conclusion, FZHY and its active compounds quercetin, baicalin and cordycepin could inhibit HBeAg production by promoting the expression of TOMM34 gene in HBV-infected hepatocytes.

Action mechanism and active components of Fuzheng Huayu Recipe against liver fibrosis via regulating macrophage with network pharmacology and transcriptomics methods

We have demonstrated that Fuzheng Huayu Recipe(FZHY) plays an anti-liver fibrosis role by regulating the polarization of intrahepatic macrophages, while the key targets in macrophages and the effective components of FZHY remain unclear. In this study, we obtained the potential anti-liver fibrosis target set of FZHY through network pharmacological analysis, and the differentially expressed gene set of FZHY for the prevention and treatment of mouse liver fibrosis through RNA-Seq of the liver tissue. The potential core targets of FZHY against liver fibrosis were obtained by degree value analysis of the common target proteins between the above two sets. Then, through the retrieval of PubMed database, we identified the potential key targets in macrophages. After that, the effective components in FZHY corresponding to key targets were obtained by reverse pharmacological analysis. Finally, we verified the regulatory effects of these effective components on the expression of key target genes by using the lipopolysaccharide-induced M1 macrophages derived from THP-1 cells. The RNA-Seq data combined with network pharmacological analysis showed that FZHY might alleviate liver fibrosis by regulating the expression of CCL2, TIMP1, and MMP2 genes in macrophages. The results of in vivo experiments showed that FZHY significantly inhibited the expression of CCL2 and TIMP1 genes and promoted the expression of MMP2 genes in liver tissues of liver fibrosis mice. The results of in vitro experiments demonstrated that FZHY and its four effective components(luteolin, ursolic acid, quercetin, and danshensu) significantly inhibited the expression of CCL2 and TIMP1 genes in M1 macrophages derived from THP-1 cells. In addition, the expression of MMP2 gene was up-regulated by luteolin, ursolic acid, and quercetin, not affected by FZHY, and down-regulated by danshensu. FZHY could inhibit the expression of CCL2 and TIMP1 genes in M1 macrophages by the four effective components to achieve the anti-inflammatory and anti-liver fibrosis effects.

An Herbal Product Alleviates Bleomycin-Induced Pulmonary Fibrosis in Mice via Regulating NF-κB/TNF-α Signaling in Macrophages.

Background and aim: Pro-inflammatory macrophages aggravated progress of pulmonary fibrosis (PF) both in patients and animal models. Fuzheng Huayu (FZHY) formula, a Chinese herbal product, is effective in treating pulmonary fibrosis in our previous study. But its action mechanism against PF relating to macrophage activation was unclear. This study was designed to evaluate the anti-fibrotic and anti-inflammatory roles of FZHY in pulmonary fibrosis and to elucidate the potential mechanisms. Methods: Network pharmacology was employed to identify the interrelationships among compounds of FZHY, potential targets and putative pathways on anti-pulmonary fibrosis. According to the data of bioinformatics analysis, the key pharmacological target for FZHY against PF was screened. The network pharmacological prediction was validated by a series of experimental assays, including CCK8, western blot and immunofluorescence staining. Then molecular mechanism of FZHY on relating to the predictive target were studied in bleomycin induced pulmonary fibrosis in mice with methylprednisolone as a positive control, and in lipopolysaccharide (LPS) stimulated cultured macrophages in culture, respectively. Results: The network pharmacology analysis reveal that a total of 12 FZHY–PF crossover proteins were filtered into a protein-protein interaction network complex and designated as the potential targets of FZHY against pulmonary fibrosis, while TNF-α signal pathway ranked at the top. FZHY and methylprednisolone could attenuate the lung fibrosis and decrease pulmonary TNF-α expression in bleomycin induced fibrotic mice, without difference between two treatments. While TNF-α was mainly originated from macrophages identified by double fluorescent staining of TNF-α and F4/80. LPS stimulated cultured macrophage polarization and activation demonstrated by the enhance contents of TNF-α and iNOS but decreased level of Arg-1. FZHY could alleviate the LPS stimulated macrophage polarization and activation demonstrated by decreasing TNF-α and iNOS and increasing Arg-1. In particular, FZHY could significantly reduce the production of p65 and the nuclear translocation of phosphorylated p65. Conclusion: Fuzheng Huayu formula has a good effect against pulmonary fibrosis induced by bleomycin in mice, whose action mechanism was associated with down-regulation of NF-κB/TNF-α signaling pathway in pro-inflammatory macrophages. These findings provided an important strategy for developing new agents against lung fibrosis and accelerated FZHY product application on patients with lung fibrosis.

Simultaneous determination of eight analytes of Fuzheng Huayu recipe in beagle dog plasma by UHPLC-Q/exactive Orbitrap HRMS and its application to toxicokinetics.

Fuzheng Huayu recipe (FZHY) is a Chinese patent medicine for the treatment of liver fibrosis. This study aimed to investigate the toxicokinetics of FZHY in beagle dogs after oral administration. Blood samples were collected on days 1, 15 and 28 after oral gavage of FZHY dosages of 400 or 1,200 mg/kg body weight once a day. A UHPLC–Q‐Orbitrap method was developed and validated to simultaneously determine and quantify eight components of FZHY in beagle dog plasma. The times to peak concentration for eight components were18–120 min. The peak concentrations (C max) of amygdalin, genistein, daidzein and 3,4‐dihydroxybenzaldehyde were 1.43–43.50 ng/ml, the areas under the concentration–time curve (AUC(0–t)) were 2.45–6,098.25 ng min/ml, and the apparent volumes of distribution (V d) were 0.05–131.23 × 104 ml/kg. The values of C max of prunasin, schisantherin A, schisandrin A and schisandrin were 7.35–1,450.73 ng/ml, the values of AUC(0–t) were 3,642.30–330,388.65 ng min/ml, and the values of V d were 11.15–1,087.18 × 104 ml/kg. No obvious accumulation of the eight compounds was observed in beagle dogs. The results showed that the method is rapid, accurate and sensitive, and is suitable for detecting the eight analytes of FZHY. This study provides an important basis for the assessment of FZHY safety.

Active Components Formulation Developed from Fuzheng Huayu Recipe for Anti-Liver Fibrosis.

To screen the active components from Fuzheng Huayu Recipe (FZHY) and redesign a new recipe composed of the active components, and validate the effect of active components formulation from FZHY against liver fibrosis. Thirty-two components from FZHY were evaluated for their activities against liver fibrosis respectively, with 6 kinds of cell models in vitro, including oxidative stressed hepatocyte in L-02, hypoxia injured/proliferative hepatic sinusoidal endothelial cells in SK-HEP-1 and human hepatic sinusoidal endothelial cells (HHSEC), and activated hepatic stellate cell in LX-2. The comprehensive activity of each component against liver fibrosis was scored according to the role of original herbs in FZHY and cell functions in fibrogenesis. Totally 7 active components were selected and combined with equal proportion to form a novel active components formulation (ACF). The efficacy of ACF on liver fibrosis were evaluated on activation of LX-2 and proliferation of HHSEC in vitro and in liver fibrosis model mice induced by dimethylnitrosamine (DMN). Totally 72 mice were divided into 6 groups using a random number table, including normal, high-dose ACF control (20 µ mol/L × 7 components/kg body weight), model, low-, medium-, high-dose ACF groups (5, 10, 20 µ mol/L × 7 components/kg body weight, respectively). Hematoxylin eosin and Sirius red stainings were used to observe inflammation and fibrosis change of liver tissue; scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to observe the effect of ACF on ultrastructure of hepatic sinusoids. Fifteen components from FZHY showed higher scores for their activity on against liver fibrosis. Among them, 7 components including tanshinone II A, salvianolic acid B, cordycepin, amygdalin, quercetin, protopanaxatriol, and schizandrin B were recombined with equal proportions to form ACF. ACF at 1,2, 4 µ mol/L showed strong inhibitory effects on activation of LX-2 and proliferation of HHSEC in vitro (all P<0.01). Compared with the model group, ACF attenuated liver collagen deposition, improved sinusoidal capillarization in a dose-dependent manner (all P<0.05). ACF exerts a satisfactory effect against experimental liver fibrosis and attenuates sinusoidal capillarization, which warrant a further research and development for herbal components formulation on liver fibrosis.

The Novel Chinese Medicine JY5 Formula Alleviates Hepatic Fibrosis by Inhibiting the Notch Signaling Pathway.

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 (CCl4)- 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 CCl4-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.

Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis

ObjectiveMicroRNAs (miRNAs) may be viable targets for treating renal interstitial fibrosis (RIF). Fuzheng Huayu recipe (FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF. MethodsRIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content, Masson’s staining and type I collagen expression were used to evaluate renal collagen deposition. Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog (PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II (Ang-II) to induce epithelial-to-mesenchymal transition (EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), while E-cadherin and α-smooth muscle actin (α-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation. ResultsFZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited α-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells. ConclusionmiR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.

Fuzheng Huayu Recipe Prevented and Treated CCl4-Induced Mice Liver Fibrosis through Regulating Polarization and Chemotaxis of Intrahepatic Macrophages via CCL2 and CX3CL1.

Background Fuzheng Huayu recipe (FZHY) is an original Chinese patent medicine which was developed and marketed by our institute. It could markedly improve liver tissue inflammation and ameliorate hepatic fibrosis in the clinical study. The intrahepatic macrophages recruitment and polarization play an important role in the progress of liver inflammation and fibrosis. Whether FZHY exerted its antiliver fibrosis effects through regulating intrahepatic macrophages phenotypic ratios is still unknown. This study aims to explore the antifibrosis mechanism of FZHY on regulating the recruitment and polarization of intrahepatic macrophages. Methods C57/B6 mice were used for the establishment of the CCl4-induced mice liver fibrosis model. Liver inflammation and fibrosis were evaluated by HE and Sirius red staining, hydroxyproline assays, and biochemical tests. The levels of chemokines and inflammatory cytokines in liver tissue were measured by RNA-Seq transcriptome analysis, western blot assay, RT-qPCR, and immunofluorescence assay. The macrophages recruitment and phenotypic polarization were observed by flow cytometry. Results FZHY significantly improved liver inflammation and reduced liver fibrosis degree. TNF signaling pathway, involved in macrophages recruitment and phenotypic polarization, was discovered by RNA-Seq transcriptome analysis. In TNF signaling pathway, CCL2 expression was significantly decreased and CX3CL1 expression was significantly upregulated by FZHY in liver tissue and primary intrahepatic macrophages. The ratio of proinflammatory hepatic resident macrophage-Kupffer cells (F4/80+CD11b−CD86+) was downregulated by FZHY, while the proportion of anti-inflammatory Kupffer cells (F4/80+CD11b−CD206+) was upregulated. Meanwhile, the ratio of proinflammatory Ly6Chigh macrophages (F4/80+CD11b+Ly6Chigh) which were recruited from blood circulation by CCL2 was reduced by FZHY, while the ratio of restorative Ly6Clow macrophages (F4/80+CD11b+Ly6Clow) which were recruited from blood circulation or induced from Ly6Chigh macrophages polarization by CX3CL1 was significantly increased. Conclusions FZHY could regulate the recruitment and polarization of intrahepatic macrophages via CCL2 and CX3CL1, so as to play its anti-inflammation and antifibrosis pharmacological effects in the liver.

Advances in anti hepatic fibrotic therapy with Traditional Chinese Medicine herbal formula.

The process of liver fibrogenesis includes a number of common and etiology-dependent or independent mechanisms and events. Up to now, there are still insufficient approved biological or chemical therapies directly targeting and reversing advanced fibrosis. The key is that once liver fibrosis is triggered, it presents a complex network control model with the activation of HSCs as the core, resulting in poor efficacy of treatment. Traditional Chinese medicine (TCM) has unique advantages in treating hepatic fibrosis because of its syndrome differentiation and treatment and comprehensive pharmacological effects of multi-channel, multi-level and multi-target. However, TCM's advantages were rarely discussed as previous reviews focused on the active ingredients of TCM and single Chinese Medicine. Therefore, this paper focuses on TCM herbal formulae's pharmacological role, target and related mechanisms in the treatment of liver fibrosis. This paper will focus on the pharmacological role, target and related mechanisms of TCM herbal formulae in the treatment of liver fibrosis. We collect English literatures or Chinese literatures with English Abstract on the treatment of liver fibrosis with TCM herbal formulae from databases including PubMed, Wiley InterScience, Science Direct OnSite/Elsevier, Ovid, Excerpta Medica Database, SpringLink, CNKI and China Biomedical Literature Database. Based on previous literatures, we summarize the TCM herbal formulae with definite anti-hepatic fibrosis effects. To some extent, classical or modern TCM herbal formulae including Yinchenhao Decoction (YCHD), Xiayuxue Decoction (XYXD), Xiaochaihutang (XCHT), Yiguanjian Decoction (YGJ), Huangqi Decoction (HQD), Dahuang Zhechong Pills (DHZC), Fuzheng Huayu Formula (FZHY), Fufang Biejia Ruangan Tablets (FFBJRG), Anluo Huaxian Pills (ALHX) and Compound 861 (Cpd861) have anti-hepatic fibrosis effect both on patients with liver fibrosis and animal models with liver fibrosis. According to the principle of syndrome differentiation and treatment, Liver fibrosis patients with different syndromes are treated with different herbal formula, which increases the difficulty of clinical efficacy research. YCHD and XYXD research lack randomized and controlled clinical trials. XCHT, YGJ and HQD research has small sample sizes despite randomized and controlled clinical trials. In contrast, most modern herbal formulae have randomized and controlled clinical trials. For instance, FZHY and ALHX recently published the research results of the combination of entecavir in the treatment of patients with chronic hepatitis B liver fibrosis or cirrhosis. Compared to anti-viral treatment with entecavir alone, this method has improved the reversion rate of liver fibrosis but still needs syndrome classification therapy of TCM. TCM Herbal formulae have a good prospect in treating liver fibrosis, but its composition of multiple drugs and a wide range of targets intensify the difficulty of studying their anti-hepatic fibrosis mechanisms. Future research needs to further study the anti-hepatic fibrosis mechanisms and select corresponding TCM herbal formula to treat patients with different syndromes of liver fibrosis or the same patient with different syndromes at different stages to achieve better curative results.

Effects of Fuzheng Huayu recipe on entecavir pharmacokinetics in normal and dimethylnitrosamine-induced hepatic fibrosis rats

Context Fuzheng Huayu recipe (FZHY) combined with entecavir (ETV) is used to treat the cirrhosis caused by chronic hepatitis B (CHB) infection. Objective To investigate the effect of FZHY on ETV pharmacokinetics under different conditions. Materials and methods A model of liver fibrosis was created by intraperitoneal injection of dimethylnitrosamine (DMN; 10 μg/kg) for 4 weeks in Wistar rats. Ultra-high-performance liquid chromatography–tandem mass spectrometry was used to determine the blood concentration of ETV. Pharmacokinetic characteristics of ETV (0.9 mg/kg) were investigated after co-administration with FZHY (0.55 g/kg) at certain time intervals in normal and model rats. Results The analytical method for ETV was validated at 0.5–50 μg/L with a correlation coefficient = 0.9996, lower limit of quantitation of 0.5 μg/L and mean accuracy of 104.18 ± 9.46%. Compared with the ETV-N group, the pharmacokinetic parameters of the EF-2 group did not change significantly, but that of the EF-0 group decreased in C max to 27.38 μg/L, in AUC0– t from 323.84 to 236.67 μg/h/L, and a delay in T max from 0.75 to 6.00 h; that of the EF-0 group presented a decrease in C max of 61.92%, delay in t 1/2 of 2.45 h and delay in T max of 2.92 h. The t 1/2e and V d/F of ETV were increased significantly to 8.01 h and 24.38 L/kg in the ETV-M group. Conclusions The effects of FZHY on ETV pharmacokinetics were diminished with an increase of interval time. The best time to administer both drugs is >2 h apart.

Study on anti- fibrotic mechanism of Fuzheng -Huayu formula to suppress autophagy in mice

Objective: To determine whether the anti-hepatic fibrosis effect of Fuzheng-Huayu formula is related to suppress autophagy in mice. Methods: C57 mice were randomly divided into normal group (N group) and model group. The model group was induced by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice, and the normal group was injected with equal volume of olive oil. After 1 week, the model group was randomly divided into model (M) group, rapamycin (Rapa) group, rapamycin plus chloroquine (Rapa+CQ) group, rapamycin plus salvianolic acid B (Rapa+Sal B) group, rapamycin plus Fuzheng -Huayu formula (Rapa+FZ) group. Each drug group was administered corresponding drugs by gavage on a daily basis, and N group and M group were given the equal amount of drinking water by gavage. After 5 weeks, the mice were sacrificed, and HE and Sirius red staining were used to observe the inflammation and collagen deposition on liver tissue in each group. The hydroxyproline content was determined by alkaline hydrolysis method. Western blotting was used to detect changes in the expression of autophagy in liver tissue and microtubule-associated protein 1 light chain 3II/I (LC3II/I), p62, α-smooth muscle actin (ɑ-SMA) and type I collagen expression. Immunofluorescence staining was used to observe the immunofluorescence localization of ɑ-SMA and LC3B in liver tissues of each group. ). A t-test was used to compare the two independent samples. LSD or Dunnett's T3 test were used to compare the mean of multiple samples. Results: There was no significant difference in N and M groups in terms of body weight. The body weight of the mice in each drug group decreased significantly (F = 14.041, P < 0.001). The liver/spleen /body weight ratios of each drug group and M group were significantly higher than the N group (F = 26.992, 6.589, P < 0.001). The expression of p62 protein in the liver tissue of mice in each drug group was lower than M group, and the difference between Rapa group and Rapa+Sal B group (F = 3.085, P = 0.039, 0.003) was statistically significant, while that of Rapa + Sal B group was lower. Compared with group M, the expression of LC3B II in Rapa group was significantly higher (F = 7.514, P = 0.01). Immunofluorescence staining showed that LC3B and α-SMA CO-stained cells were absent in the liver of mice in N group, and co-stained cells were found in the liver of mice in M group. The co-stained cells in the liver of mice in each drug group were significantly higher than M group, and the co-stained cells in Rapa+FZ group were fewer. Compared with the N group, the collagen deposition of M group and each drug group was significantly increased; the collagen deposition of each drug group was lower than that of the M group. There was no statistically significant difference between each drug group. Compared with N group (77.75 + 48.79), hydroxyproline in liver tissue of mice in M group was significantly increased (293.48 + 84.43) (F = 3.015, P = 0.005), and the content of hydroxyproline in liver tissue of mice in each drug group was lower than M group, but the difference was not statistically significant (F = 0.750, P = 0.573). Compared with the N group, the expressions of α-SMA and type I collagen in the M group were significantly increased (F = 27.718, 18.893, P < 0.01). The expression of α-SMA in Rapa group and Rapa+Sal B group was similar to M group, while Rapa + CQ group and Rapa + FZ group were significantly lower than Rapa group and M group (P < 0.01). The expression of type I collagen in Rapa + CQ group was significantly higher than Rapa group (P = 0.017), while the expression of type I collagen in Rapa + FZ group was significantly lower than M group (P = 0.013). Conclusion: Autophagy of hepatic stellate cells was observed in carbon tetrachloride-induced liver fibrosis model. Rapamycin can promote autophagy in hepatocytes and hepatic stellate cells. Fuzheng-Huayu formula and Salvianolic Acid B might antagonize the effect of rapamycin on autophagy.

Metabolic mechanisms of Fuzheng-Huayu formula against liver fibrosis in rats

Ethnopharmacological relevanceFuzheng-Huayu formula (FZHY) is traditionally used to treat liver fibrosis in clinic. The study was conducted to investigate the metabolic mechanisms of FZHY against liver fibrosis in rats. Materials and methodsRats with CCl4 -induced liver fibrosis were treated with FZHY and its components, including amygdalin, cordyceps polysaccharide and gypenoside, respecitively. Liver fibrosis and function were assesed by histopathological examination, Western blot and serum biochemical detection. Metabolic profiling of liver tissue, serum and urine in each group were detected by gas chromatography-mass spectrometry (GC-MS) and transcriptomic changes were tested by gene chip. RT-qPCR was used to validate levels of different expressed genes (DEGs) with statistical significance. Metabolic network together with DEGs was constructed based on KEGG database. ResultsFZHY effectively improved liver fibrosis better than the mixture or single use of gypenoside, cordyceps sinensis mycelia and amygdalin. FZHY treatment widely modulated the metabolic profiles perturbed by liver fibrosis, involving several important metabolic pathways, including glycolysis/gluconeogenesis, glucose-alanine cycle, citrate cycle, galactose metabolism, tryptophan metabolism, urea cycle, etc. It also increased alanine and decreased glucose levels in liver tissue and decreased both of them in serum and urine, which were dysregulated by CCl4 treatment. Additionally, FZHY also upregulated expression of metabolic enzymes including Hk2, Adh1 and Gpt increased, and downregulated Gs and Acss2. ConclusionFZHY improved liver fibrosis in rats via altering the metabolic pathways and regulating gene expression of involved metabolic enzymes.

Active Compounds Derived from Fuzheng Huayu Formula Protect Hepatic Parenchymal Cells from Apoptosis Based on Network Pharmacology and Transcriptomic Analysis.

Fuzheng huayu formula (FZHY), an antifibrotic traditional Chinese medicine, is frequently used for the treatment of liver fibrosis. In this study, network analysis, transcriptomic analysis, assays of cell apoptosis, viability and protein expression were used for investigating the effects and mechanisms of compounds derived from FZHY on hepatic parenchymal cell (HPC) protection and hepatic stellate cell activation. Network pharmacology analysis found that 6 major compounds and 39 potential targets were important network nodes. Our analysis predicted that the active compounds of FZHY, including hederagenin, luteolin and tanshinone IIA inhibited cell apoptosis (p < 0.05), increased PI3K expression and reduced cleaved caspase 3 expression and the Bax/Bcl-w ratio (p < 0.05) in L02 cells that had apoptosis induced by TNF-α. Few significant changes caused by FZHY, hederagenin, luteolin and tanshinone IIA were observed in hepatic stellate Lx2 cells upon TGF-β1 induction. These data suggest that FZHY is active against liver fibrosis, protects hepatic parenchymal cells from apoptosis, and recovers liver function, possibly through the effects of its active compounds hederagenin, luteolin and tanshinone IIA and is involved in the inhibition of apoptosis in HPCs, possibly through regulating the PI3K, ERK, cleaved caspase 3 and Bax/Bcl-w levels.

The Active Components of Fuzheng Huayu Formula and Their Potential Mechanism of Action in Inhibiting the Hepatic Stellate Cells Viability - A Network Pharmacology and Transcriptomics Approach.

Purpose: This study aimed to identify the active components of Fuzheng Huayu (FZHY) formula and the mechanism by which they inhibit the viability of hepatic stellate cells (HSCs) by a combination of network pharmacology and transcriptomics.Methods: The active components of FZHY formula were screened out by text mining. Similarity match and molecular docking were used to predict the target proteins of these compounds. We then searched the STRING database to analyze the key enriched processes, pathways and related diseases of these target proteins. The relevant networks were constructed by Cytoscape. A network analysis method was established by integrating data from above network pharmacology with known transcriptomics analysis of quiescent HSCs-activated HSCs to identify the most possible targets of the active components in FZHY formula. A cell-based assay (LX-2 and T6 cells) and surface plasmon resonance (SPR) analysis were used to validate the most possible active component-target protein interactions (CTPIs).Results: 40 active ingredients in FZHY formula and their 79 potential target proteins were identified by network pharmacology approach. Further network analysis reduced the 79 potential target proteins to 31, which were considered more likely to be the target proteins of the active components in FZHY formula. In addition, further enrichment analysis of 31 target proteins indicated that the HIF-1, PI3K-Akt, FoxO, and chemokine signaling pathways may be the primary pathways regulated by FZHY formula in inhibiting the HSCs viability for the treatment of liver fibrosis. Of the 31 target proteins, peroxisome proliferator activator receptor gamma (PPARG) was selected for validation by experiments at the cellular and molecular level. The results demonstrated that schisandrin B, salvianolic acid A and kaempferol could directly bind to PPARG, decreasing the viability of HSCs (T6 cells and LX-2 cells) and exerting anti-fibrosis effects.Conclusion: The active ingredients of FZHY formula were successfully identified and the mechanisms by which they inhibit HSC viability determined, using network pharmacology and transcriptomics. This work is expected to benefit the clinical application of this formula.

A metabolic mechanism analysis of Fuzheng-Huayu formula for improving liver cirrhosis with traditional Chinese medicine syndromes.

Fuzheng-Huayu formula (FZHY), a Chinese herbal mixture prescription, has been proven effective in treating liver fibrosis and cirrhosis in both clinical trials and animal experiments. In this study we assessed the metabolic mechanisms of traditional Chinese medicine (TCM) syndrome-based FZHY treatment in liver cirrhosis (LC). A total of 113 participants, including 50 healthy controls and 63 LC patients, were recruited. According to the diagnosis and differentiation of the TCM syndromes, the LC patients were classified into 5 TCM syndrome groups including the liver stagnation syndrome (LSS), spleen deficiency and damp overabundance syndrome (SDDOS), damp-heat accumulation syndrome (DHAS), liver-kidney Yin deficiency syndrome (LKYDS), and blood stagnation syndrome (BSS), and administered FZHY for 6 months. FZHY treatment significantly decreased serum levels of hyaluronic acid (HA), a biochemical marker for LC, as well as TCM syndrome scores (the TCM syndrome scores were decreased in all the groups with significant decreases in the LSS and LKYDS groups). Furthermore, FZHY treatment gradually shifted the metabolic profiles of LC patients from a pathologic state to a healthy state, especially in LC patients with LSS and LKYDS. Twenty-two differently altered metabolites (DAMs) were identified, including carbohydrates, amino acids, fatty acids, etc with 9 DAMs in LSS patients, 9 in LKYDS patients, and 4 in other patients. The metabolic pathways involved in the conversion of amino acids and the body's detoxification process were regulated first, followed by the pathways involved in the body's energy supply process. In conclusion, the evaluation of the effect of TCM syndrome-based FZHY treatment show that FZHY has a better effect on LKYDS and LSS than on the other TCM syndromes, and the metabolic mechanisms might be involved in the increased detoxification function in LKYDS and the improvement of energy supply in LSS, which provides important evidence for the clinical application of TCM syndrome-based treatment.