Yin-chen Wu-ling powder alleviate cholestatic liver disease: Network pharmacological analysis and experimental validation.

BACKGROUND: Yin-chen Wu-ling Powder (YWP) has potential therapeutic effects on cholestatic liver disease (CLD), however, its active compounds and conceivable mechanism are as yet indistinct. METHODS: The network pharmacology and gene function annotation examined the multiple active ingredients, potential targets, and possible mechanisms of YWP in CLD treatment. Then the molecular docking reassured the reliability of the core compounds including the key genes and farnesoid X receptor (FXR). Finally, The Mdr2-/- mice were used to test the effect and mechanism of YWP against CLD. RESULTS: The network analysis identified nine main active ingredients, including quercetin, capillarisin, eupalitin, isorhamnetin, skrofulein, genkwanin, cerevisterol, gederagenin, and sitosterol. The PPI network predicted the ten hub genes involved were AKT1, MAPK1, MAPK14, IL6, RXRA, ESR1, IL10, NCOA1, CAV1, and EGFR. The KEGG and GO analysis showed that YWP might contribute to CLD treatment through the PI3K/Akt and MAKP signalings to manage pathological reactions, for instance, inflammatory responses. The molecular docking displayed a functional similarity among the core compounds with ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) on the effects on AKT1, MAPK1, MAPK14, RXRA, and ESR, and the affinity to FXR. In addition, the YWP could significantly attenuate hepatic injury and improve inflammatory response in Mdr2-/- mice. The mechanism exploration showed that YWP mainly decreased inflammatory response by inhibiting AKT/P38MAPK signaling. CONCLUSION: This study firstly revealed the multiple active ingredients, potential targets, and possible mechanism of YWP to treat CLD based on network pharmacology Analysis and molecular docking. YWP could alleviate cholestasis in Mdr2-/- mice by impairing inflammation via inhibiting AKT/P38MAPK Signaling.

Bushen Jianpi Formula Combined with Entecavir for the Treatment of HBeAg-Negative Chronic Hepatitis B: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Bushen Jianpi formula (BSJPF, also known as Lingmao formula) is a traditional Chinese medicine for chronic hepatitis B (CHB). The previous study has suggested that the treatment combination of BSJPF and entecavir (ETV) can achieve a significant loss of hepatitis B e antigen (HBeAg) and a significant decrease in serum level of hepatitis B virus (HBV) DNA in HBeAg-positive CHB patients with mildly elevated alanine aminotransferase. Objective: This study aimed to evaluate the efficacy and safety of BSJPF combined with ETV for treating HBeAg-negative CHB patients. Methods: A total of 640 patients were assigned randomly to the treatment group (receiving BSJPF combined with ETV for 96 weeks) or the control group (receiving a placebo combined with ETV for 96 weeks) in a 1 : 1 ratio. The primary endpoints are the rate of loss of hepatitis B surface antigen (HBsAg). The secondary outcomes included the rate of decrease in the HBsAg concentration to ≥1 lg·IU/mL, the HBV DNA suppression, the decline of the level of covalently closed circular DNA (cccDNA) in the liver, histological improvements, and the rate of ALT normalization. Results: The rate of HBsAg loss in the treatment group was significantly higher than that of the control group (5.5% versus 1.8%, P=0.031). There were 11.1% of patients in the treatment group who recorded a reduction in HBsAg ≥1 lg·IU/mL, which is better than 5.9% of patients in the control group (P=0.043). There was no significant difference between the two groups with regard to the rate of HBV DNA clearance, the reduction in intrahepatic cccDNA, and the rate of ALT normalization (P > 0.05). The rate of liver fibrosis improvement in the treatment group was better than that of the control group (35.5% versus 11.8%, P=0.031), but there was no difference in necroinflammatory improvement (P > 0.05). The adverse events (AEs) were similar between the two groups, except for the abnormal kidney function, with 2.2% in the control group and 0.0% in the treatment group (P=0.028). Conclusion: The combination of BSJPF and ETV can increase the rate of HBsAg loss and the rate of histological fibrosis improvement without serious adverse events in CHB patients. Trial Registration. This trial is registered with ChiCTR-IOR-16009880 on November 16, 2016-retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=16836.

Long-Term Traditional Chinese Medicine Combined with NA Antiviral Therapy on Cirrhosis Incidence in Chronic Hepatitis B Patients in the Real-World Setting: A Retrospective Study.

OBJECTIVE: To evaluate the impact of long-term Traditional Chinese Medicine (TCM) syndrome differentiation combined with antiviral therapy with Nucleos (t) ide analogues (NAs) on the incidence of cirrhosis in patients with chronic hepatitis B. METHODS: This retrospective cohort study included 521 patients with chronic hepatitis B who underwent a treatment course of ≥3 years from 1998-2019. Of the 521 patients, 261 were defined as TCM users while 260 were TCM nonusers (control group). All the enrolled subjects were followed up until February 2019 to measure the incidence and hazard ratio (HR) of cirrhosis, and the Cox proportional hazards regression model was used to analyze the independent factors affecting the occurrence of cirrhosis. RESULTS: The cumulative incidence of TCM users and nonusers was 6.9% and 13.5%, respectively (P=0.013). Results of the Kaplan-Meier analysis demonstrated that TCM users had a significantly lower cumulative incidence of cirrhosis than TCM nonusers (P=0.011), and TCM users had a significantly lower liver cirrhosis risk than TCM nonusers (adjusted HR = 0.416, 95% CI, 0.231-0.749). The histological evaluation revealed improved fibrosis in 45.0% of TCM users and 11.1% of TCM nonusers (P=0.033). The analysation of the prescriptions including total 119 single Chinese herbs medicinal demonstrated that "replenish qi and fortify the spleen," "clear heat and dispel dampness," and "soothe the liver and regulate qi" are the main treatment methods of TCM for CHB. CONCLUSIONS: Our study demonstrated that long-term TCM use may attenuate liver cirrhosis risk in patients with chronic hepatitis B (CHB).

RPB5-Mediating Protein Promotes Cholangiocarcinoma Tumorigenesis and Drug Resistance by Competing With NRF2 for KEAP1 Binding.

BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.

Expression of IL-26 predicts prognosis of patients with hepatocellular carcinoma after surgical resection.

BACKGROUND: There is no data regarding prognostic impact of interleukin (IL)-26 on outcomes of patients with hepatocellular carcinoma (HCC). The present study aimed to evaluate the prognostic impact of IL-26 on HCC patients undergoing liver resection. METHODS: From 2003 to 2008, 122 patients with HCC who received surgical curative resection were enrolled. Patients were stratified into IL-26-upper and -lower groups according to the median expression level from immunohistochemical staining of resected specimens. Prognostic impact of IL-26 was estimated using Kaplan-Meier curves. Univariate and multivariate analyses were performed to evaluate time-dependent prognostic impact and independency of IL-26. Demographic and clinical factors that were associated with IL-26 were comprehensively identified. RESULTS: Prognosis of the patients with high level of IL-26 revealed to be significantly unfavorable in both cumulative recurrence-free survival (P < 0.001) and overall survival (P = 0.002). Upper expression of IL-26 (HR: 1.643; 95% CI: 1.021 to 2.644; P = 0.041) and microvascular invasion (HR: 3.303; 95% CI: 1.255 to 8.696; P = 0.016) were identified as significant independent prognostic factors for overall survival in the multivariable analysis. CONCLUSIONS: IL-26 is a novel prognostic factor for HCC after resection. Evaluation of IL-26 expression may be potentially valuable in clinical therapy when planning individualized follow-up schedule and evaluating candidates for prophylactic adjuvant treatment to prevent recurrence.

Tumor-associated lymphangiogenesis predicts unfavorable prognosis of intrahepatic cholangiocarcinoma.

BACKGROUND: Tumor-associated lymphangiogenesis is considered significant in number of solid malignancies. However, its impact on prognosis of intrahepatic cholangiocarcinoma (ICC) after resection remains further confirmation. Herein, we conducted this study to evaluate prognostic impact of tumor-associated lymphangiogenesis in patients with ICC. METHODS: Extent of tumor-associated lymphangiogenesis of ICC was evaluated by quantifying microlymphatic vessel density (MLVD) from immunohistochemical staining of a lymphatic endothelial-specific antibody (podoplanin). Clinicopathological characteristics were comprehensively analyzed to identify MLVD-associated factors. The patients were stratified into high and low MLVD groups according to the distinctive correlation between the MLVD and overall survival using the Spearman's correlation test. Kaplan-Meier estimation was performed to confirm prognostic impact of MLVD in patients with ICC. Univariate and multivariate analyses were performed using the Cox proportional hazard model. RESULTS: The MLVD between 4 to 12 counts showed inverse proportion to the overall survival (Spearman's r = - 0.66; 95% confidence interval [CI], - 0.82 to - 0.39; p <  0.0001), which was set as a cut-off for the high MLVD group, whereas the MLVD between 13 to 25 showed no correlation to the overall survival (r = - 0.11; 95% CI, - 0.38 to 0.19; p = 0.4791). The high MLVD group showed more frequent lymph node metastasis (p <  0.001) and were more likely to suffer from recurrence of the tumor compared to the low MLVD group (p <  0.001). The high MLVD was found to be independently associated with reduced overall and recurrence-free survival. The 5-year overall survival of the patients with high MLVD was significantly lower compared to those with low MLVD (0% vs 48%). CONCLUSIONS: Our study reveals that tumor-associated lymphangiogenesis is significantly associated with increased lymphatic metastasis, recurrence of the tumor, and reduced overall survival in patients with ICC, thus providing guidance when estimating postresection prognosis.

Expression of VEGFR-3 in intrahepatic cholangiocarcinoma correlates with unfavorable prognosis through lymphangiogenesis.

Background & aims: VEGFR-3 has been shown of great significance in lymph node metastasis and some malignancies, however, its expression in tumors and impact on outcome of intrahepatic cholangiocarcinoma (iCCA) remains unknown. The aim of this study was to assess the role of VEGFR-3 positive tumors for prognosis of iCCA and tumor-associated lymphangiogenesis. Methods: Clinicopathological features, prognostic factors and survival rate were analyzed to evaluate the influence of VEGFR-3 positive expression on prognosis of iCCA. In addition, tumor-associated lymphangiogenesis quantified as micro-lymphatic vessel density (MLVD) was assessed to explore the correlation between VEGFR-3 expression and lymph node metastasis for iCCA. Results: Patients with VEGFR-3 positive tumors had increased lymph node metastasis (p=0.025) and were more likely to suffer from tumor recurrence compared with VEGFR-3 negative tumors (p<0.001). VEGFR-3 expression in tumors was identified as an independent prognostic factor for both overall and recurrence-free survival in surgical resected patients with iCCA. In addition, higher MLVD was significantly associated with VEGFR-3 positive expression in tumors (p<0.001), which facilitate lymph node metastasis and significantly worse survival rates. Conclusions: Our study reveals that VEGFR-3 positive expression in tumors represents an independent prognostic factor for both overall and recurrence-free survival in hepatic resected patients with iCCA. VEGFR-3 positive tumors favor lymph node metastasis, tumor recurrence and worse outcomes through tumor-associated lymphangiogenesis.

IFN-γ inhibits liver progenitor cell proliferation in HBV-infected patients and in 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-fed mice.

BACKGROUND & AIMS: Proliferation of liver progenitor cells (LPCs) is associated with inflammation and fibrosis in chronic liver diseases. However, how inflammation and fibrosis affect LPCs remains obscure. METHODS: We examined the role of interferon (IFN)-γ, an important pro-inflammatory and anti-fibrotic cytokine, in LPC expansion in HBV-infected patients and in mice challenged with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- or choline-deficient, ethionine-supplemented (CDE) diet as well as in primary LPCs and LPC cell line. RESULTS: The CK19 staining scores correlated with inflammation and fibrosis grades in the livers from 110 HBV-infected patients. Nine-month IFN-γ treatment decreased LPC numbers, inflammation, and fibrosis in these HBV-infected patients. Similarly, a two-week IFN-γ treatment also decreased LPC activation in DDC-treated mice. Disruption of IFN-γ or its signaling components (e.g., IFNGR, STAT1, and IRF-1) increased LPC proliferation and liver fibrosis in DDC-fed mice. In contrast, deletion of IFN-γ did not increase, but rather slightly reduced LPC proliferation in CDE-fed mice. In vitro, IFN-γ attenuated proliferation of the LPC cell line BMOL and of primary LPCs from wild type mice, but not STAT1(-/-) or IRF-1(-/-) mice. Furthermore, co-culture assays suggest that IFN-γ can indirectly promote LPC proliferation via the activation of macrophages but attenuate it via the inhibition of hepatic stellate cells. CONCLUSIONS: IFN-γ inhibits LPC expansion via the direct inhibition of LPC proliferation and indirect attenuation of liver fibrosis in the DDC model, but it may also enhance LPC expansion via the promotion of inflammation in the CDE model; thereby playing dual roles in regulating LPC proliferation in vivo.

Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells.

Adult hepatic progenitor (oval) cells are facultative stem cells in liver, which participate in a range of human liver diseases, including hepatocellular carcinoma (HCC). However, the molecular pathways regulating the expansion and differentiation of these cells are poorly understood. We show that active Wnt/beta-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active beta-catenin mutant promotes expansion of the oval cell population in the regenerated liver. More importantly, we identify a subpopulation of less differentiated progenitor-like cells in HCC cell lines and primary HCC tissues, which are defined by expression of the hepatic progenitor marker OV6 and endowed with endogenously active Wnt/beta-catenin signaling. These OV6(+) HCC cells possess a greater ability to form tumor in vivo and show a substantial resistance to standard chemotherapy compared with OV6(-) tumor cells. The fraction of tumor cells expressing OV6 is enriched after Wnt pathway activation, whereas inhibition of beta-catenin signaling leads to a decrease in the proportion of OV6(+) cells. In addition, the chemoresistance of OV6(+) HCC progenitor-like cells can be reversed by lentivirus-delivered stable expression of microRNA targeting beta-catenin. These results highlight the importance of the Wnt/beta-catenin pathway in activation and expansion of oval cells in normal rodent models and human HCCs. OV6(+) tumor cells may represent the cellular population that confers HCC chemoresistance, and therapies targeted to the Wnt/beta-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy.

Signal regulatory protein alpha negatively regulates both TLR3 and cytoplasmic pathways in type I interferon induction.

Recognition of double-stranded RNA (dsRNA) activates interferon-regulatory factor 3 (IRF3)-dependent expression of anti-viral factors. The innate immune system recognizes viral dsRNA through two distinct pathways. First, the Toll-like receptor 3 (TLR3) detects dsRNA phagocytosed in endosomes. In addition, the helicases retinoic acid induced protein I (RIG-I)/melanoma differentiation associated gene 5 (MDA5) binds cytoplasmic dsRNA generated during viral replication. Both RIG-I/MDA5 and TLR3 can bind polyriboinosinic:polyribocytidylic acid (poly(I:C)), the synthetic analog of viral dsRNA, and mediate type I IFN production. Here we show that signal regulatory protein (SIRP) alpha negatively regulates both TLR3- and RIG-1/MDA5-dependent anti-viral pathways. Suppression of SIRPalpha expression by RNA interference results in enhanced activation of IRF3 and MAPK pathways after poly(I:C) treatment, coupled with the up-regulation of IFN-beta and IFN-beta-inducible gene transcriptional activation. The requirement of phosphoinositide 3-kinase (PI3K) activity for the induction of IFN-beta and IFN-beta-inducible genes by dsRNA is supported by the observation that a PI3K inhibitor failed to activate IFN-beta and IFN-beta-inducible gene expression. PI3K, whose activity is essential for activation of IRF3, is recruited to the phosphorylated tyrosine residues of SIRPalpha upon poly(I:C) stimulation, which lead to a reduction in the activity of the downstream kinase AKT. Thus SIRPalpha may accomplish its inhibitory function in type I IFN induction, in part, through its association and sequestration of the signal transducer PI3K.

LPS-induced down-regulation of signal regulatory protein {alpha} contributes to innate immune activation in macrophages.

Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor kappaB (NF-kappaB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) alpha, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-kappaB signaling pathways. In addition, SIRPalpha can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPalpha in innate immunity. We provide evidences that SIRPalpha is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPalpha expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPalpha expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPalpha reduced macrophage responses to LPS. Knockdown of SIRPalpha caused prolonged activation of MAPKs and NF-kappaB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPalpha-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPalpha may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPalpha functions as a biologically important modulator of TLR signaling and innate immunity.