Changes in the total volume and contour density of oval cells in hepatic lobules of rats with carbon tetrachloride-induced hepatic fibrosis： A stereological study / 临床肝胆病杂志

ObjectiveTo quantitatively investigate the changes in the total volume and contour density of hepatic oval cells （HOC） in hepatic lobules of rats with carbon tetrachloride （CCl4）-induced hepatic fibrosis. MethodsA total of 11 healthy male Sprague-Dawley rats were randomly divided into control group with 5 rats and hepatic fibrosis group with 6 rats， and CCl4 and olive oil suspension were injected subcutaneously twice a week， 3 mL/kg each time. After five weeks of hepatic fibrosis modeling， five liver tissue blocks with a size of about 1 mm3 were randomly selected from the liver of each rat to prepare one Epon812 epoxy resin-embedded ultrathin section， and the stereological method and transmission electron microscopy were used for the quantitative analysis of the total volume and contour density of HOC in the hepatic lobules of rats. In addition， four liver tissue blocks with a thickness of 2 mm were randomly selected from the remaining liver of each rat to prepare two paraffin-embedded Masson staining sections， and the degree of liver fibrosis in each rat was qualitatively evaluated according to the Metavir staging criteria for liver fibrosis. The independent-samples t test was used for comparison of continuous data between groups. ResultsThe quantitative stereological analysis showed that the total volume of HOC in hepatic lobules was 15.40±7.63 mm3 in the control group and 146.80±114.00 mm3 in the liver fibrosis group， and compared with the control group， the total volume of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 8.53 times （t=-2.551， P=0.031）； the contour density of HOC in hepatic lobules was 56.20±40.40 in the control group and 566.50±317.00 in the liver fibrosis group， and compared with the control group， the contour density of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 9.08 times （t=-3.539， P=0.006）. Qualitative observation showed that liver fibrosis stage of rats reached stage Ⅱ-Ⅲ according to the Metavir scoring criteria， and massive proliferation of HOC was observed around the proliferation site of hepatic stellate cells in the perisinusoidal space of rats. ConclusionCCl4 induces significant proliferation of HOC in hepatic lobules of rats with liver fibrosis.

Stellate Ganglion Block as an Adjunctive Intervention for Chronic Subjective Tinnitus： Efficacy and Predictive Indicators / 中山大学学报(医学科学版)

ObjectiveTo explore the efficacy and predictive indicators of stellate ganglion block （SGB） as an adjunctive intervention for chronic subjective tinnitus and accumulate experience for the application of SGB in the clinical treatment of tinnitus. MethodsA retrospective review was conducted on the data of chronic subjective tinnitus patients who received SGB intervention， with unsatisfactory outcomes otherwise. Pure tone audiometry （PTA）， tinnitus loudness evaluation and Pittsburgh sleep quality index （PSQI） were used. The tinnitus handicap inventory （THI） scores were compared before and after SGB intervention. Correlation analysis and linear regression equations were employed to identify the potential indicators predicting the effectiveness of SGB intervention. Statistical analysis was performed by SPSS 24.0 software. ResultsBy April 2023， a total of 107 patients with chronic subjective tinnitus had undergone SGB intervention， including 67 male and 40 female， with a mean age of （45.32±11.40） years old and an average tinnitus history of （20.32±24.64） months ［16 （12~20）］. Only 7 patients （6.54%） quitted the intervention for personal reasons， which demonstrated good compliance with the intervention. No patients experienced adverse reactions such as infection at the injection site， hematoma， nerve injury， local anesthetic intoxication and so on， which revealed good safety. After SGB intervention， THI scores decreased to below 36 points in 77 patients and decrease by 10 points or more in 12 of the remaining patients， with a total effective rate of 89%. A paired sample t-test showed a significant difference in THI scores before and after SGB intervention （t=15.575， P<0.001）， indicating good improvement. Pearson correlation analysis suggested that pre-intervention THI scores and subjective tinnitus loudness were significantly positively correlated with the improvement level of THI scores （P<0.05）. Further stepwise linear regression analysis found that "pre-intervention THI scores" had statistical significance （P<0.001）， with a regression coefficient of 0.308， predicting a 17.4% improvement level in THI scores. ConclusionsDue to its good and safe short-term effects， SGB intervention can be used as a supplementary option for chronic subjective tinnitus when other interventions are not ideal， especially for patients with higher THI scores. However， further research is needed to clarify the long-term efficacy and underlying mechanisms， in order to establish a more solid theoretical basis for SGB intervention in the treatment of subjective tinnitus.

The regulatory effect of morphine postconditioning in left stellate ganglion on remodeling after myocardial infarction / 中国药理学通报

Aim To investigate the regulatory effect of morphine postconditioning in the LSG on remodeling after myocardial infarction. Methods SD rats were randomly divided into four groups: sham operation group (Sham), myocardial infarction group (MI), myocardial infarction + saline group (Control) and myocardial infarction + morphine postconditioning group (MI + Morphine) . The rat MI model was constructed by ligating the left anterior descending coronary artery, and then morphine was given to the LSG by percutaneous posterior approach. After four weeks, the changes of cardiac function in rats were detected by ultrasound. Masson staining was used to detect fibrosis changes; the expression of Collagen I and Collagen III protein was detected by Western blot. The mRNA expression of ANP and BNP was detected by RT-qPCR. The expression of JJLOR in LSG was detected by immunofluorescence. The concentration of catecholamine in plasma and myocardial tissue was detected by ELISA. Results Compared with the sham group, the cardiac function of the MI group was significantly impaired, the myocardial tissue showed significant fibrosis changes, and the concentration of catecholamine in plasma and myocardial tissue significantly increased. Compared with the control group, the MI + Morphine group reduced myocardial fibrosis collagen deposition in rats after MI, inhibited the expression of ANP and BNP in myocardial tissue, reduced the concentration of catecholamine, and improved the cardiac function of MI rats. Immunofluorescence results showed that JJLOR was expressed in LSG after MI and increased after morphine postconditioning. Conclusions This study shows that morphine postconditioning in the LSG has a protective effect on myocardial remodeling after myocardial infarction. The mechanism may be related to the activation of JJLOR in the LSG by morphine and the reduction of catecholamine release from sympathetic nerve endings.

Establishment and genotype identification of hepatic stellate cell-specific Grk2 gene knockout mouse model / 中国药理学通报

Aim To establish a stable hepatic stellate cell ( HSC ) -specific G protein-coupled receptor kinase 2 ( GRK2 ) knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC. Methods The loxP-labeled Grk2 gene mouse (Grk2

Role of podoplanin in hepatic stellate cell activation and liver fibrosis / 临床肝胆病杂志

ObjectiveTo investigate the role and mechanism of podoplanin （PDPN） in hepatic stellate cell （HSC） activation and liver fibrosis. MethodsLiver biopsy samples were collected from 75 patients with chronic hepatitis B who attended Department of Infectious Diseases， Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， for the first time from September 2019 to June 2022， and RT-PCR and immunohistochemistry were used to measure the expression of PDPN in liver tissue of patients in different stages of liver fibrosis. A total of 12 male C57/BL6 mice were randomly divided into control group and model group. The mice in the model group were given intraperitoneal injection of 10% CCl4， and those in the control group were injected with an equal volume of olive oil， for 6 weeks. HE staining and Sirius Red staining were used to observe liver histopathological changes； primary mouse liver cells were separated to measure the mRNA expression of PDPN in various types of cells； primary mouse HSCs were treated with PDPN protein， followed by treatment with the NF-‍κB inhibitor BAY11-708， to measure the expression of inflammatory factors in HSCs induced by PDPN. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Spearman correlation analysis was used to investigate data correlation. ResultsAs for the liver biopsy samples， there was a relatively low mRNA expression level of PDPN in normal liver， and there was a significant increase in the mRNA expression level of PDPN in liver tissue of stage S3 or S4 fibrosis （all P<0.001）. Immunohistochemical staining showed that PDPN was mainly expressed in the fibrous septum and the hepatic sinusoid， and the PDPN-positive area in S4 liver tissue was significantly higher than that in S0 liver tissue （t=8.892， P=0.001）. In normal mice， PDPN was mainly expressed in the hepatic sinusoid， and there was a significant increase in the expression of PDPN in CCl4 model mice （t=0.95， P<0.001）， mainly in the fibrous septum. RT-PCR showed a significant increase in the mRNA expression of PDPN in the CCl4 model mice （t=11.25， P=0.002）. Compared with hepatocytes， HSCs， Kupffer cells， and bile duct endothelial cells， hepatic sinusoidal endothelial cells showed a significantly high expression level of PDPN （F=20.56， P<0.001）. Compared with the control group， the primary mouse HSCs treated by PDPN protein for 15 minutes showed significant increases in the mRNA expression levels of the inflammation-related factors TNFα， CCL3， CXCL1， and CXCR1 （all P<0.05）， and there were significant reductions in the levels of these indicators after treatment with BAY11-7082 （all P<0.05）. ConclusionThere is an increase in the expression of PDPN mainly in hepatic sinusoidal endothelial cells during liver fibrosis， and PDPN regulates HSC activation and promotes the progression of liver fibrosis via the NF-κB signaling pathway.

Overview and prospects of an in vitro cell model for studying liver fibrosis / 中华肝脏病杂志

Liver fibrosis incidence and adverse outcomes are high; however, there are no known chemical drugs or biological agents that are specific and effective for treatment. The paucity of a robust and realistic in vitro model for liver fibrosis is one of the major causes hindering anti-liver fibrosis drug development. This article summarizes the latest progress in the development of in vitro cell models for liver fibrosis, with a focus based on the analysis of induction and activation of hepatic stellate cells, cell co-culture, and 3D model co-construction, as well as concurrent potential methods based on hepatic sinusoidal endothelial cell establishment.

Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Effects of specific knockdown of ribosomal protein S5 in hepatic stellate cells on liver fibrosis / 药学实践杂志

Objective To observe the effect of specific knockdown of hepatic stellate cells (HSC) ribosomal protein S5 (RPS5) on liver fibrosis in rats. Methods The glial fibrillary acidic protein (GFAP) promoter-driven RPS5 shRNA adenovirus was established, and AdGFa2-shRPS5 and its control AdGFa2 shNC were used to transfect primary rat HSCs and hepatocytes, respectively. RPS5 was determined by Western-blot and Real Time PCR, α-SMA and type I collagen expression; the rat liver fibrosis model was established by dimethyl nitrosamine (DMN) and bile duct ligation (BDL), and intrahepatic HSC was specifically knocked down by tail vein injection of adenovirus of RPS5 levels. The pathological changes of liver tissue sections were analyzed by HE staining; the content of hydroxyproline, sections of Sirius red and Masson staining were used to evaluate collagen deposition; immunohistochemical staining was used to detect the expression of α-SMA and RPS5. Results AdGFa2-shRPS5 specifically knocked down the expression level of RPS5 in HSC and increased the expression of α-SMA and type I collagen in vitro. The in vivo results showed that in two animal models of chronic liver injury, specific knockdown of RPS5 expression in HSCs promoted HSC activation, increased the deposition of extracellular matrix, and promoted liver fibrosis. Conclusion RPS5 is essential for HSC activation and liver fibrosis, which could be a potential target for the treatment of liver fibrosis.

Huangqi Decoction, a compound Chinese herbal medicine, inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-β axis / 中西医结合学报

OBJECTIVE@#Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis.@*METHODS@#The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting.@*RESULTS@#Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment.@*CONCLUSION@#Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

Role and mechanism of action of the Mongolian medicine / 临床肝胆病杂志

Objective To investigate the role and mechanism of action of Scabiosa atropurea in inhibiting the proliferation of hepatic stellate cells using cell experiment. Methods A total of 20 Wistar rats were randomly divided into control group and administration group, with 10 rats in each group. The rats in the control group were given normal saline by gavage, and those in the administration group were given Scabiosa atropurea by gavage to prepare drug-containing serum. HSC-T6 cells were incubated with the serum from the control group (10%) or the low-, middle-, and high-dose serum containing Scabiosa atropurea (10%, 15%, and 20%, respectively). MTT assay was used to observe the effect of different drug concentrations on cells in different periods of time; flow cytometry was used to measure cell apoptosis; qRT-PCR and Western blot were used to measure the mRNA and protein expression levels of fibrosis markers (α-SMA, collagen Ⅰ) and PI3K/Akt signaling pathway-related factors in hepatic stellate cells (HSCs). A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t - test was used for further comparison between two groups. Results Compared with the control group, the low-, middle-, and high-dose serum containing Scabiosa atropurea groups had a significant reduction in the OD value of cells (all P < 0.05) and a significant increase in the overall apoptosis rate of cells (all P < 0.05). The results of qRT-PCR showed that compared with the control group, the low-, middle-, and high-dose serum containing Scabiosa atropurea groups had significant reductions in the mRNA expression levels of α-SMA, collagen Ⅰ, PI3K, and Akt and a significant increase in the mRNA expression level of PTEN (all P < 0.05); Western blot showed that compared with the control group, the low-, middle-, and high-dose serum containing Scabiosa atropurea groups had significant reductions in the protein expression levels of α-SMA, collagen Ⅰ, PI3K, Akt, and p-Akt and a significant increase in the protein expression level of PTEN (all P < 0.05). Conclusion The Mongolian medicine Scabiosa atropurea can inhibit the proliferation of HSC-T6 cells and promote their apoptosis, possibly by regulating fibrosis markers and the PI3K/Akt signaling pathway to exert an anti-liver fibrosis effect.

Research advances in the reversal of liver fibrosis / 临床肝胆病杂志

Chronic liver injury caused by any etiology will lead to liver fibrosis, and it was believed in the past that liver fibrosis is a static and irreversible pathophysiological process. In recent years, with the rapid development of molecular biology and the in-depth research on the microscopic aspect of the liver, more and more evidence has shown that liver fibrosis is a dynamic and reversible process. This article reviews the reports of different methods for evaluating the reversal of liver fibrosis caused by various etiologies, summarizes the pathogenesis and reversal mechanism of liver fibrosis, reviews the therapeutic drugs for reversal, and summarizes the current evaluation methods for liver fibrosis, and finally, it is believed that timely clearance or control of potential etiology may help to achieve the reversal of liver fibrosis to a certain degree.

Association between liver sinusoidal endothelial cells and liver fibrosis / 临床肝胆病杂志

Liver sinusoidal endothelial cells (LSECs) are crucial to the maintenance of hepatic homeostasis under physiologic conditions, while under the conditions of pathological liver damage, LSEC can respond to the damage by changing their structure through the process called capillarization, thereby aggravating liver damage. In addition, the interaction between LSEC and other cells in the liver plays a certain role in the development and progression of liver fibrosis, especially the interaction between LSEC and hepatic stellate cells, which are the primary effector cells of liver fibrosis. This article mainly elaborates on the role of LSEC in the development and progression of liver fibrosis during chronic liver injury.

Non-coding RNA-mediated TGF-β1/Smads Signaling Pathway and Liver Fibrosis， and Intervention by Active Components of Chinese Medicinal： A Review / 中国实验方剂学杂志

Liver fibrosis is a wound healing response that occurs in the setting of chronic liver injury and is caused by imbalance in the synthesis and degradation of extracellular matrix （ECM）. If left untreated， it can progress to liver cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cell （HSC） is now well established as a central driver of liver fibrosis. The activated HSC will transform into myofibroblasts that produce ECM protein. Transforming growth factor-β1 （TGF-β1） can induce the activation of hepatic stellate cell （HSC）， and TGF-β1/Smads signaling pathway is one of the important pathways to promote liver fibrosis. Non-coding RNA （ncRNA） does not encode proteins during the transcription but plays an important regulatory role in the post-transcriptional process of genes. Accumulating evidence shows that the occurrence of liver fibrosis is closely related to the abnormal expression of ncRNA which participates in the activation of HSC by regulating TGF-β1 signal transduction and then affects the process of liver fibrosis. MiRNA-mediated TGF-β1/Smads signaling pathway can not only promote liver fibrosis but also play a role in anti-fibrosis. Long non-coding RNA （lncRNA） not only promotes the development of liver fibrosis by binding to target genes but also enhances TGF-β1 signal transduction by acting as competitive endogenous RNA. circular RNA （circRNA） acts as a ''sponge'' to regulate TGF-β1/Smads pathway， thereby inhibiting HSC activation and exerting the anti-liver fibrosis effect. Chinese medicinal plays an essential part in the prevention and treatment of liver fibrosis， and the active components can inhibit TGF-β1/Smads pathway by regulating the expression of miRNA， thus alleviating liver fibrosis. This article reviews the role and mechanism of miRNA-， lncRNA- and circRNA-mediated TGF-β1/Smads signaling pathway in liver fibrosis and summarizes the anti-liver fibrosis effect of active components of Chinese medicinals by regulating miRNA-mediated TGF-β1/Smads signaling pathway， which can serve as a reference for clinical treatment of liver fibrosis and the development of new drugs.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro / 中国血吸虫病防治杂志

Objective To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. Methods The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. Results The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. Conclusion rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling / 中国结合医学杂志

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.

Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway / 中国中药杂志

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Establishment of a novel co-cultured liver cancer model based on tumor microenvironment / 中华肿瘤杂志

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.

Therapeutic effect of Rab11 inhibitor cyclin-dependent kinase inhibitor-73 on liver fibrosis and its related mechanisms / 中华肝胆外科杂志

Objective:To investigate the therapeutic effect and potential molecular mechanisms of cyclin-dependent kinase inhibitor-73 (CDKI-73), the Rab11 inhibitor, on liver fibrosis.Methods:Human LX2 cells were divided into four groups: negative control group, transforming growth factor-β (TGF-β) group, CDKI-73 group and TGF-β+ CDKI-73 group. Fifteen 5-week-old female C57 mice with body weight of (18.04±0.62) g were divided into 3 groups with 5 mice in each group: control group (intraperitoneal injection of olive oil + vehicle gavage), carbon tetrachloride (CCl 4) group (intraperitoneal injection of CCl 4 + vehicle gavage) and CCl 4+ CDKI-73 group (intraperitoneal injection of CCl 4+ CDKI-73 gavage). Another 15 5-week-old female C57 mice with body weight of (18.06±0.34) g were divided into 3 groups with 5 mice in each group: sham operation group (Sham), bile duct ligation (BDL) group + vehicle group (BDL+ vehicle gavage) and bile duct ligation+ CDKI-73 group (BDL+ CDKI-73 gavage). The expression of α-smooth muscle actin (α-SMA) and fibronectin(FN)in LX2 cells were analyzed by Western blot. Masson and Sirius red were used to examine the liver fibrosis after CDKI-73 treatment in vivo. Immunohistochemistry (IHC) was utilized to examine the expression of α-SMA in mice liver. Results:Collagen content assessed by Sirius red and Masson staining and α-SMA expression evaluated by IHC were all increased in CCl 4 group compared with control group ( q=38.47, 24.99, 36.79). Moreover, the collagen content and α-SMA expression in CCl 4 + CDKI-73 treatment group were obviously decreased compared with CCl 4 group ( q=24.72, 14.87, 27.50), and the differences were statistically significant (all P<0.001). Compared with Sham group, collagen content and α-SMA expression in bile duct ligation group were increased ( q=28.23, 41.01, 44.16). Furthermore, in BDL group, after treatment with CDKI-73, the collagen content and α-SMA expression were notably decreased ( q=22.88, 34.31 and 33.97, all P<0.001). Consistent with in vivo results, the relative expression levels of α-SMA and FN protein in TGF-β group were higher than those in TGF-β+ CDKI-73 group (α-SMA: 3.71±0.34 vs. 1.28±0.31; FN: 3.21±0.39 vs. 0.83±0.06, all P<0.001). The mRNA relative expression levels of α-SMA and FN in TGF-β group were higher than those in TGF-β+ CDKI-73 group, and the differences were statistically significant ( P<0.001). However, the relative expression of TGF-β receptor Ⅱ protein in CDKI-73 group was higher than those in negative control group (4.68±0.63 vs. 1.00±0.22, P=0.004). The relative expression level of phosphorylated SMAD2 in TGF-β+ CDKI-73 group was lower than those in TGF-β group (1.67±0.24 vs. 3.99±0.44, P<0.001). Transwell assay showed that 0.5 μmol/L CDKI-73 could effectively inhibit the migration of LX2 cells, and the inhibitory ability became stronger with the increase of CDKI-73 concentration. Conclusion:CDKI-73 can inhibit the activation of hepatic stellate cells and liver fibrosis by inhibiting Rab11-dependent TGF-β signaling pathway both in vivo and in vitro.