摘要
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.
摘要
ObjectiveTo investigate the effect of the protein kinase RNA-like endoplasmic reticulum kinase （PERK）/eukaryotic initiation factor 2α （eIF2α） pathway in endoplasmic reticulum stress on the activation of hepatic stellate cells （HSC）. MethodsPathological sections of normal liver tissue after surgery were collected from 11 patients with hepatic fibrosis （S1-S4） and 9 patients with hepatic hemangioma and hepatic adenoma confirmed by liver biopsy， and immunohistochemistry was used to measure the protein expression levels of PERK， eIF2α， and C/EBP homologous protein （CHOP）. Human HSC-LX2 cells were treated with different concentrations of the endoplasmic reticulum stress inducer thapsigargin （0， 125， 250， 500， and 1 000 nmol/L）， and qRT-PCR was used to measure the mRNA expression level of PERK， while Western blot was used to measure the protein expression levels of PERK， inositol requiring protein 1 （IRE1）， activating transcription factor 6 （ATF6）， CHOP， and α-smooth muscle actin （α-SMA）. The method of lentivirus transfection was used to construct a PERK stable overexpression LX-2 group and a control group； qRT-PCR was used to measure the mRNA expression levels of PERK， eIF2α， and α-SMA， Western blot was used to measure the protein expression levels of PERK， phosphorylated eIF2α （p-eIF2α）， and α-SMA， and immunofluorescence assay was used to measure the expression of collagen type I alpha 1 （COL1A1）. 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 Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. ResultsCompared with normal liver tissue， the liver tissue of patients with hepatic fibrosis had significantly higher expression levels of PERK， eIF2α， and CHOP （Z=-3.56， t=-5.75， Z=-3.52， all P<0.001）. Compared with the solvent group， the groups treated with different concentrations of thapsigargin had significant increases in the expression levels of the endoplasmic reticulum-associated proteins PERK， CHOP， IRE1， ATF6， and α-SMA （all P<0.05）. Compared with the control group， the PERK stable overexpression group had significant increases in the mRNA expression levels of PERK， eIF2α， and α-SMA and the protein expression levels of PERK， p-eIF2α， and α-SMA （all P<0.05）， and immunofluorescence assay showed a significant increase in the expression level of COL1A1 in the PERK stable overexpression group （P<0.05）. ConclusionPERK overexpression can induce the expression of α-SMA and COL1A1 in LX-2 cells， suggesting that the PERK signaling pathway might be one of the important mechanisms of HSC activation.
摘要
Objective @# To explore the role of long non-coding RNA XR_378418 (LncRNA XR_378418) in the bi- ological behavior of hepatic stellate cells line JS-1 and to probe the potential molecular mechanism of LncRNA XR _378418 involved in liver fibrosis based on transcriptome sequencing．@*Methods @#In this study，the recombinant plasmid of pcDNA-LncRNA XR_378418 and control plasmid pcDNA-NC were constructed and transfected into JS-1 cells respectively．Then，the expression level of LncRNA XR_378418 was analyzed by quantitative real-time PCR (RT-qPCR) ．The effect of overexpression of LncRNA XR_378418 on proliferation and migration of JS-1 cells were detected by cell counting kit-8 assay ( CCK-8 ) and scratch assay，respectively． Finally，by high-throughput se- quencing analysis，the effect of XR_378418 on the transcriptomics of JS-1 cells was analyzed. @*Results @#T-qPCR results showed that the expression level of LncRNA XR_378418 in the overexpression group was significantly higher than that in the control group (P＜0. 05) ．The results of CCK-8 and scratch experiment suggested that the prolifer- ation and migration in the pcDNA-LncRNA XR _ 378418 group significantly increased． Furthermore ，the high- throughput sequencing analysis showed that a total of 248 genes were screened by gene differential analysis ，of which 127 were up-regulated ，and 117 were down-regulated． Gene Ontology ( GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that LncRNA XR_378418 could regulate cell adhesion，autophagy and Ca2 + signaling，etc．@*Conclusion @#LncRNA XR_378418 promotes the proliferation and migration of JS-1 cells and affects the expression of genes related to cell adhesion and calcium signaling in JS-1 cells.
摘要
ObjectiveTo investigate the effect of oxaliplatin on the activation of hepatic stellate cells （HSCs）， as well as the association of oxaliplatin with microRNA-30a-5p and autophagy. MethodsHSC-LX2 cells were cultured and divided into groups according to the following three protocols： control group， PDGF treatment group， oxaliplatin treatment group， oxaliplatin+PDGF treatment group； control group， microRNA-30a-5p transfection group， PDGF treatment group， microRNA-30a-5p transfection+PDGF treatment group； control group， 3-MA group， microRNA-30a-5p inhibitor group， microRNA-30a-5p inhibitor+3-MA group. Western Blot was used to measure the expression of HSC activation-related proteins （Collagen-I and alpha-smooth muscle actin ［α- SMA］） and HSC autophagy-related proteins （Beclin-1， P62， and LC3B）； LysoTracker staining and immunofluorescence assay were used to measure the expression of LC3B autophagosomes； RT-PCR was used to measure the expression level of microRNA-30a-5p； bioinformatics techniques were used to predict the potential targets of microRNA-30a-5p in HSCs. 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. ResultsAfter the cells were treated with oxaliplatin， RT-PCR results showed that the oxaliplatin treatment group had a significantly higher expression level of microRNA-30a-5p than the control group （P<0.01）； Western Blot showed that the oxaliplatin treatment group had significant reductions in the expression levels of the HSC activation-related proteins α-SMA and Collagen-Ⅰ and the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （all P<0.001）； immunofluorescence assay showed that the oxaliplatin treatment group had a significantly lower number of autophagosomes than the control group （P<0.05）. After HSC-LX2 cells were transfected with microRNA-30a-5p mimic， compared with the control group， the microRNA-30a-5p mimic group had significant reductions in the expression levels of the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （P<0.05） and the HSC activation-related protein Collagen-Ⅰ （P<0.001）； after HSC-LX2 cells were transfected with microRNA-30a-5p inhibitor， Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC activation-related proteins Collagen-Ⅰ and α-SMA and the autophagy-related protein Beclin 1 （t=2.41， 2.32， and 4.57， all P<0.05）. Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC autophagy-related protein Beclin 1 and the HSC activation-related protein α-SMA （both P<0.05）， and after the treatment with the autophagy inhibitor 3-MA， there were no significant differences in the expression of these proteins between the two groups （P>0.05）. The bioinformatics analysis using TargetScan， PicTar， and miRanda databases showed that the autophagy-related protein Beclin-1 might be a potential target of miRNA-30a-5p. ConclusionOxaliplatin can inhibit the activation of HSCs by upregulating the expression of microRNA-30a-5p， which provides new ideas and a new target for the treatment of liver fibrosis.
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Objective To explore the role and potential mechanisms of all-trans retinoic acid （ATRA） on activation and oxidative stress of hepatic stellate cell （HSC）. Methods Platelet-derived growth factor （PDGF-bb, 10 ng/ml） was applied to induce the activation of HSCs, which was then treated with ATRA at a dosage of 5 μmol/L for 48 h. The effects of ATRA on HSC activation were evaluated by detecting changes in cell growth viability and phenotypic marker expression. The effects of ATRA on HSC oxidative stress were evaluated by detecting changes in intracellular reactive oxygen species （ROS）, reduced glutathione （GSH） and malondialdehyde （MDA）, and the expression of antioxidant genes. The effects of ATRA on HSC autophagic activity were evaluated by detecting changes in autophagy marker expression and autophagic flow. Results Compared with the PDGF-bb group, the cell viability was significantly reduced in ATRA-treated HSCs （P<0.01）, as well as the expression of α-SMA and Collagen I. The intracellular levels of ROS and MDA were significantly reduced in ATRA-treated HSCs （P<0.01）, whereas the GSH level was significantly increased （P<0.01）. The expression levels of antioxidant genes （NRF2, HO-1, and ATF4）, were significantly higher in ATRA-treated HSCs than those in the normal ones under PDGF-bb condition （P<0.01）. Meanwhile, the expression of autophagy markers Beclin 1 and LC3 Ⅱ/I, and signal of autophagy flow in ATRA-treated HSCs were found to be significantly reduced （P<0.01）. Conclusion ATRA significantly inhibited PDGF-bb-induced HSC activation and reduced the level of oxidative stress and autophagic activity of HSCs, which had potential applications in the prevention and treatment of liver fibrosis.
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ObjectiveTo investigate the regulatory effect of miRNA-933 on the apoptosis and proliferation of human hepatic stellate cell line LX-2 and its mechanism. MethodsFirstly， with human liver tissue for research， gene microarray technology was used to detect the differentially expressed genes in liver tissue between liver cirrhosis/chronic hepatitis B tissue and normal liver tissue， among which the significantly differentially expressed miRNAs were identified， and thus miRNA-933 was determined as the research object. Then， with the human hepatic stellate cell line LX-2 for research， miRNA-933 mimic and inhibitor （miRNA-933 siRNA） were used to construct the LX-2 models of overexpression and knockdown， and the cells transfected with mimic-NC （overexpression） or siRNA-NC （knockdown） were established as the negative control group. Quantitative real-time PCR and Western blot were used to measure the expression levels of miRNA-933 and activation biomarkers； techniques such as cell proliferation assay and flow cytometry were used to investigate the effect and mechanism of miRNA-933 on cell apoptosis， proliferation， and activation. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and Bonferroni correction was also performed. ResultsA total of 18 significantly differentially expressed miRNAs were obtained based on the results of gene microarray， among which miRNA-933 was significantly downregulated （P<0.05）. After LX-2 cells were transfected with miRNA-933 mimic or siRNA， compared with the negative control group， miRNA-933 siRNA significantly downregulated the expression of miRNA-933 （P=0.000 7）， while miRNA-933 mimic significantly upregulated the expression of miRNA-933 （P=0.000 3）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly upregulated the expression of collagen I and α-SMA （P<0.001）， while miRNA-933 mimic significantly inhibited the expression of collagen I and α-SMA （P<0.05）. Flow cytometry showed that compared with the negative control group， miRNA-933 siRNA significantly downregulated the apoptosis rate of LX-2 cells （P=0.031 9）， and miRNA-933 mimic significantly upregulated the apoptosis rate of LX-2 cells （P=0.005 5）. Western blot showed that compared with the negative control group， miRNA-933 siRNA could inhibit the expression of Caspase-3 （P=0.006 7） and poly（ADP-ribose） polymerase-1 （PARP-1） （P=0.003 0） and upregulate the expression of B-cell lymphoma-2 （Bcl-2） in LX-2 cells （P=0.002 0）， while miRNA-933 mimic could significantly upregulate the expression of Caspase-3 （P=0.011 8） and PARP-1 （P=0.049 5） and downregulated the expression of Bcl-2 （P=0.002 1）. Cell proliferation assay showed that compared with the negative control group， miRNA-933 siRNA could promote the proliferation of LX-2 cells （P=0.011 5）， while on the contrary， miRNA-933 mimic could inhibit the proliferation of LX-2 cells （P=0.001 2）. Western blot and quantitative real-time PCR showed that miRNA-933 siRNA significantly inhibited the expression of Kruppel-like factor 6 （KLF6） and downregulated the expression of activating transcription factor 4 （ATF4）， activating transcription factor 3 （ATF3）， and C/EBP homologous protein （CHOP）， while miRNA-933 mimic promoted the expression of the above proteins （all P<0.05）. ConclusionThis study shows that miRNA-933 may promote cell apoptosis and inhibit cell activation and proliferation by promoting the activation of the KLF6/ATF4/ATF3/CHOP/Bcl-2 signal axis in LX-2 cells.
摘要
Objective To investigate the effect of NaAsO2 on ferroptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were cultured in vitro,and different concentrations of NaAsO2(5μmol/L,10μmol/L,15μmol/L)were infected with LX-2 cells for 24h in a group design to construct the activation model of LX-2 induced by NaAsO2 in vitro,and a control group was set up.Mitochon-drial structure of LX-2 cells treated with NaAsO2 was observed by transmission electron microscopy(TEM).Fe2+levels were detected by fluorescence microscope and fluorescent enzyme label.The content of malondialdehyde(MDA)was determined by the colorimetric meth-od.The protein expression levels of SLC7A11,GPX4,and α-smooth muscle actin(α-SMA)were detected by Western blot.Results TEM showed that mitochondrial membrane integrity was damaged and mitochondrial ridges were reduced and disappeared in the NaAsO2group.In addition,compared with the control group,Fe2+levels in NaAsO2 treatment groups were increased(P＜0.05).There were statistically significant differences in MDA content among different doses of NaAsO2groups(F=7.18,P＜0.05).Compared with the control group,MDA content of LX-2 cells in 5 and 15μmol/L NaAsO2groups was higher than that in the control group(P＜0.05).At the translation level,the expression of fibrosis index α-SMA protein level was up-regulated with the increase of NaAsO2dose(P＜0.05),the protein expression levels of ferroptosis index SLC7A11 and GPX4decreased in a dose-dependent manner with the increase of the dose of NaAsO2(P＜0.05).Conclusion Ferroptosis is involved in the activation of LX-2 cells induced by NaAsO2.
摘要
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.
摘要
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.
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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.
Subject(s)
Humans , Hepatic Stellate Cells/radiation effects , Interleukin-6 , NF-kappa B , NF-KappaB Inhibitor alpha , Phenotype , RNA , RNA, Circular/metabolism , Tumor Necrosis Factor-alpha , ELAV-Like Protein 1/metabolism摘要
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.
Subject(s)
Humans , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1/metabolism , RNA, Long Noncoding/pharmacology , Drugs, Chinese Herbal/pharmacology , MicroRNAs/genetics , Hepatic Stellate Cells/pathology , Liver Cirrhosis/metabolism , Cell Proliferation , Transforming Growth Factors/pharmacology摘要
OBJECTIVE To study the inhibitory effects and possible mechanism of naringenin on the activation of hepatic stellate cells. METHODS Using human hepatocytes LO2 as reference， based on drug intervention concentration screened by MTT assay， the effects of naringenin （Western blot assay and trypan blue staining test in 10， 20， 40 μmol/L， immunofluorescence assay in 40 μmol/L） on the expressions of liver fibrosis markers protein （collagen Ⅰ， α-SMA） and mRNA （α1-pro collagen Ⅰ， α-SMA） in human hepatic stellate cells LX2， and the expressions of cell apoptosis and apoptosis-related proteins （Bcl-2， Bax， cleaved caspase-3） were investigated. The apoptosis agents （Z-VAD-FMK， FMK）， ferroptosis pathway inhibitor ferrostatin-1， and programmed death pathway inhibitor necrostatin-1 were used to verify the mechanism of the above effects. RESULTS The naringenin could significantly down-regulate protein expressions of collagen Ⅰ （except for naringenin 10 μmol/L） and α-SMA， mRNA expressions of α1-pro collagen Ⅰ （except for naringenin 10 μmol/L） and α-SMA （P＜0.05）； it also induced LX2 cell apoptosis and increased its apoptotic ratio， down-regulated the protein expression of Bcl-2 while up-regulated the protein expressions of Bax （except for naringenin 10 μmol/L） and cleaved caspase-3 （except for naringenin 10 μmol/L）. FMK could reverse above effects of naringenin on LX2 cells （P＜0.05）. CONCLUSIONS Naringenin can inhibit the activation of hepatic stellate cells LX2 through activating the cell apoptosis signal， which plays ameliorative role in liver fibrosis.
摘要
Hepatic fibrosis is a pathological reparative response of the liver to chronic injury and a crucial step in the progression of chronic liver disease， characterized mainly by the activation of hepatic stellate cells and diffuse deposition of extracellular matrix. Currently， there is no ideal specific drug for the treatment of liver fibrosis in clinical practice. In recent years， with the development and progress of traditional Chinese medicine （TCM） in the treatment of liver fibrosis， TCM has been widely recognized for its significant therapeutic effect and fewer adverse reactions. The phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway is an important pathway that affects the formation and development of liver fibrosis. It mainly plays a role in liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells， promoting their apoptosis， reducing oxidative stress in liver cells， decreasing the deposition of extracellular matrix， and enhancing liver cell autophagy. This article summarized the mechanisms by which Chinese medicinal monomers regulated the PI3K/Akt pathway to exert their effects on liver fibrosis and their synergistic effects with other signaling pathways， providing a theoretical basis and references for the development of new drugs for the treatment of liver fibrosis with TCM.
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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.
摘要
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.
Subject(s)
NF-kappa B/metabolism , Hepatic Stellate Cells , Transforming Growth Factor beta1/metabolism , NF-KappaB Inhibitor alpha/metabolism , Intercellular Adhesion Molecule-1/metabolism , Isodon , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Toll-Like Receptor 4/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Lipopolysaccharides/pharmacology , Signal Transduction , Colchicine/pharmacology , Caspases摘要
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.
Subject(s)
Male , Mice , Animals , Keratin-18 , Actins , Desmin , Liver , Hepatocytes , Hepatic Stellate Cells摘要
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.
Subject(s)
Humans , Liver Cirrhosis/pathology , Hepatic Stellate Cells , Cell Culture Techniques , Endothelial Cells摘要
OBJECTIVE@#To observe the effect of exosomes secreted by lipopolysaccharides (LPS)-stimulated macrophages on hepatic stellate cell activation and migration and explore the underlying molecular mechanism.@*METHODS@#Human monocyte THP-1 cells were induced to differentiate into macrophages using propylene glycol methyl ether acetic acid (PMA, 100 ng/mL, 24 h) followed by stimulation with LPS, and the culture supernatant of macrophages was collected for extraction of the exosomes by ultracentrifugation. The expression of miR-155-5p in the exosomes was detected using qRT-PCR. A Transwell co-culture system was used to observe the effects of the macrophage-derived exosomes on LX2 cell (a hepatic stellate cell line) proliferation, migration, oxidative stress and the expression of fibrosis biomarkers, which were also observed in LX2 cells transfected with miR-155-5p-mimics or miR-155-5p-inhibitors. Western blotting was used to detect the expressions of SOCS1 and its downstream signal pathway proteins.@*RESULTS@#Treatment with the exosomes from LPS-stimulated macrophages significantly enhanced the proliferation and migration ability of LX2 cells and increased the levels of oxidative stress and expressions of the fibrosis markers such as type Ⅰ collagen (P < 0.05). The expression of miR-155-5p in the exosomes secreted by macrophages was significantly increased after LPS treatment (P < 0.01). LX2 cells overexpressing miR-155-5p also exhibited significantly enhanced proliferation and migration with increased oxidative stress levels and expression of type Ⅰ collagen (P < 0.05), and interference of miR-155-5p expression produced the opposite effects. Western blotting showed that miR-155-5p overexpression obviously inhibited SOCS1 expression and promoted p-Smad2/3, Smad2/3 and RhoA protein expressions in LX2 cells (P < 0.05).@*CONCLUSION@#LPS stimulation of the macrophages increases miR-155-5p expression in the exosomes to promote activation and migration and increase oxidative stress and collagen production in hepatic stellate cells.
Subject(s)
Humans , Hepatic Stellate Cells , Lipopolysaccharides/pharmacology , Collagen Type I , Exosomes , Macrophages , MicroRNAs摘要
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.
Subject(s)
Rats , Male , Mice , Animals , Transforming Growth Factor beta/metabolism , Amygdalin/therapeutic use , Endothelial Cells/metabolism , Olive Oil/therapeutic use , Rats, Wistar , Smad Proteins/metabolism , Liver Cirrhosis/metabolism , Liver , Transforming Growth Factor beta1/metabolism , Signal Transduction , Collagen Type I/metabolism , Carbon Tetrachloride , Hepatic Stellate Cells摘要
Objective To explore the extraction and purification method of Kupffer and hepatic stellate cells from mouse liver and provide references and suggestions for the separation and extraction method ology of primary non-parenchymal cells from mouse liver.Methods After in vivo collagenase perfusion digestion,various reagents and method,such as Percoll and OptiPrep,were used to extract C57BL/6 mice Kupffer and hepatic stellate cells,and evaluate their purity by flow cytometry and immunofluorescence.Results The two-layer Percoll method to extract Kupffer cells and the two-layer OptiPrep method to extract hepatic stellate cells were feasible,and purity reached>90%.The cell yield was 1～2×107/liver,and the cell survival rate was>90%.After 48 hours of primary cell culture,the number of F4/80-positive Kupffer cells and α-SMA-positive hepatic stellate cells reached>90%.Conclusions The separation and extraction method of Kupffer and hepatic stellate cells from mouse liver are perfect,reliable,cost-effective,and reproducible.