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ABSTRACT BACKGROUND: The effect of weight loss (WL) on histopathological aspects of non-alcoholic fatty liver disease (NAFLD) may provide further insights into the dynamics of hepatic recovery after WL. OBJECTIVE: To analyze the effects of pre-operative WL on insulin resistance- and NAFLD-related histology in individuals undergoing bariatric surgery (BS) with or without pre-operative WL. DESIGN AND SETTING: A matched cross-sectional study was conducted at a public university hospital and a private clinic in Campinas, Brazil. METHODS: An analytical, observational, cross-sectional study was conducted using prospectively collected databases of individuals who underwent BS and liver biopsy at either a public tertiary university hospital (with pre-operative WL) or a private clinic (without pre-operative WL). Random electronic matching by gender, age, and body mass index (BMI) was performed and two paired groups of 24 individuals each were selected. RESULTS: Of the 48 participants, 75% were female. The mean age was 37.4 ± 9.6. The mean BMI was 38.9 ± 2.6 kg/m2. Fibrosis was the most common histopathological abnormality (91.7%). Glucose was significantly lower in the WL group (92 ± 19.1 versus 111.8 ± 35.4 mg/dL; P = 0.02). Significantly lower frequencies of macrovesicular steatosis (58.3% versus 95.8%; P = 0.004), microvesicular steatosis (12.5% versus 87.5%; P < 0.001), and portal inflammation (50% versus 87.5%; P = 0.011) were observed in the WL group. CONCLUSION: Pre-operative WL was significantly associated with lower frequencies of macro- and mi- crovesicular steatosis, portal inflammation, and lower glycemia, indicating an association between the recent trajectory of body weight and histological aspects of NAFLD.
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ObjectiveTo explore the mechanism and pathway of Gandou Fumu decoction （GDFMD） in the development of liver fibrosis in Wilson's disease （WD）. MethodFirst， 30 TX-j mice were randomly divided into the model group， high-dose， medium-dose， and low-dose GDFMD groups， and penicillamine group， with six mice in each group， and another six wild-type mice were used as the normal group. The high-dose， medium-dose， and low-dose GDFMD groups were intragastrically administered drugs of 13.92， 6.96， 3.48 g·kg-1. In the penicillamine group， 0.1 g·kg-1 of penicillamine was given by intragastric administration. The model group and the normal group were given equal volume of normal saline， once a day， for four consecutive weeks. Samples were collected four weeks after gavage， and enzyme-linked immunosorbent assay （ELISA） was used to detect type Ⅲ procollagen peptide （PCⅢ）， collagen type Ⅳ （Col Ⅳ）， hyaluronic acid （HA）， and laminin （LN）. Hematoxylin-eosin （HE）， Masson， and picric acid-Sirus red collagen （Sirus Red） staining were used to observe the histopathological changes of liver fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， immunohistochemistry， and Western blot were used to observe the expressions of α-smooth muscle actin （α-SMA） and collagen type Ⅰ （Col Ⅰ）， which were related to the activation of hepatic stellate cells （HSCs）. The expression of miR-29b-3p was observed by Real-time PCR. The expression of Unc-51-like kinase 1 （ULK1） and its downstream-related factors were observed by Western blot. The downstream genes of miR-29b-3p were verified by the dual luciferase reporter gene detection method. ResultCompared with the normal group， the four items of liver fibrosis （PCⅢ， Col Ⅳ， HA， and LN） in the model group were significantly abnormal （P<0.01）， and the pathology was significantly abnormal. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was up-regulated （P<0.05， P<0.01）， and miR-29b-3p expression was down-regulated （P<0.01）. ULK1， p-ULK1， autophagy-related gene 13 （Atg13）， p-Atg13， Beclin-1， FAK family kinase-interacting protein of 200 kDa （FIP200）， activating molecule in BECN1-regulated autophagy protein 1 （AMBKA1）， and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ） were up-regulated （P<0.05， P<0.01）. p62 protein expression was down-regulated （P<0.01）. Compared with the model group， the four items of liver fibrosis in the high-dose， medium-dose， and low-dose GDFMD groups and the penicillamine group were significantly improve （P<0.01）， and the pathological conditions were improved. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was down-regulated （P<0.05， P<0.01）， and the expression of miR-29b-3p was up-regulated （P<0.01）. ULK1， p-ULK1， Atg13， p-Atg13， Beclin-1， FIP200， AMBKA1， and LC3Ⅱ/Ⅰ were down-regulated （P<0.05， P<0.01）， and p62 protein expression was up-regulated （P<0.01）. The prediction software predicted that there was a binding site between miR-29b-3p and ULK1. The dual-luciferase reporter gene detection method indicated that the luciferase activity of the ULK1-WT plasmid-transfected cell group was reduced when miR-29b-3p mimics were co-cultured （P<0.01）. ConclusionGDFMD can regulate ULK1-mediated autophagy by up-regulating miR-29b-3p and further exert its anti-hepatic fibrosis effect in Wilson's disease.
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Resumen Antecedentes: El estándar de oro para determinar el grado de fibrosis hepática (FH) continúa siendo la evaluación de la biopsia. Existen técnicas de morfometría que permiten cuantificar la FH en estudios histopatológicos. Objetivo: Medir la correlación entre la evaluación histológica de FH y la cuantificación por morfometría del porcentaje de fibrosis (PF) mediante HepaScan. Material y métodos: Estudio piloto observacional, analítico, transversal, prospectivo y prolectivo en el que se analizaron cortes histopatológicos de hígado de 29 personas fallecidas por alguna hepatopatía y 22 personas fallecidas por otras causas (controles). El PF se calculó con HepaScan en fotografías digitales de cortes histológicos teñidos con la técnica Masson, comparándolo con el diagnóstico de tres patólogos expertos. Resultados: Fueron analizadas 401 imágenes del grupo con hepatopatía y 250 del grupo de control. La concordancia interobservador tuvo un índice kappa de 0.329. Entre los grupos de clasificación histopatológica existieron diferencias estadísticas en el PF (p = 0.0001). La capacidad predictiva de HepaScan con base en el área bajo la curva característica operativa del receptor fue de 0.983, 0.812 y 0.895 para fibrosis leve, moderada y severa, respectivamente. Conclusiones: HepaScan mostró muy buen desempeño para evaluar el PF en cortes histológicos, por lo que puede coadyuvar al diagnóstico patológico cualitativo.
Abstract Background: The gold standard for determining the degree of liver fibrosis (LF) continues to be biopsy evaluation. There are morphometry techniques that allow LF to be quantified on histopathological studies. Objective: To measure the correlation between LF histological evaluation and fibrosis percentage (FP) morphometric quantification using the HepaScan software. Material and methods: Observational, analytical, cross-sectional, prospective, prolective pilot study in which liver histological sections from 29 people who died from some liver disease and from 22 people who died from other causes (controls) were analyzed. FP was calculated with HepaScan on digital photographs of histological sections stained with the Masson technique, comparing it with the diagnosis established by three expert pathologists. Results: Four-hundred and one images from the group with liver disease and 250 from the control group were analyzed. Inter-observer agreement had a kappa index of 0.329. There were FP statistically significant differences (p = 0.0001) between histopathological classification groups. HepaScan predictive capacity based on the area under the receiver operating characteristic curve was 0.983, 0.812, and 0.895 for mild, moderate, and severe fibrosis, respectively. Conclusions: HepaScan showed very good performance for evaluating FP in histological sections, which is why it can contribute to qualitative pathological diagnosis.
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Objective To explore the possible mechanism of Radix Paeoniae Alba on hepatic fibrosis based on network pharmacology. Methods Tcmsp database was used to screen the active components of Paeonia alba. With the help of PubChem and Swiss target prediction database, the potential action targets of the effective components of Paeonia Alba were predicted. GeneCards and OMIM databases were used to screen the corresponding targets of liver fibrosis, and venn2.1.0 was used to obtain the common targets of white peony and liver fibrosis. Cytoscape 3.9.0 software was used to build the network diagram of “white peony - active ingredients - intersection target - liver fibrosis” and to predict the main active sites. String database was used to draw the PPI network. Go analysis of effective targets and enrichment analysis of KEGG in pathway were performed by David database. Results Six effective components, 213 targets of Paeonia Alba and 155 hepatic fibrosis targets were screened. There were 49 targets of Radix Paeoniae Alba in the treatment of liver fibrosis. The main active ingredients are kaempferol, paeoniflorin, mairin and β-Sitosterol. Go enrichment analysis showed 269 biological processes, 30 cell compositions, 64 molecular functions, and 67 pathways in KEGG pathway enrichment analysis. Conclusion The mechanism of anti-hepatic fibrosis of Radix Paeoniae Alba has been preliminarily studied through network pharmacology, which shows that Radix Paeoniae Alba has multi-component, multi-target, and multi-channel effects, and provides reference for further experimental research.
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Objectives@#This study aimed to compare the severity of COVID-19, inflammatory parameters and clinical outcomes among patients with normal and subnormal levels of Vitamin D.@*Methodology@# This is a retrospective cohort study of 135 patients admitted in a tertiary hospital for COVID-19. Patients were grouped according to their Vitamin D level. Primary outcome measure was the composite of all-cause mortality and morbidity. Other outcome measures determined were the comparison among the groups on the severity of COVID-19 infection, changes in inflammatory parameters, length of hospital stay and duration of respiratory support.@*Results@#There was a significant trend of higher ICU admission, mortality (p-value= 0.006) and poor clinical outcome (p-value=0.009) among the Vitamin D deficient group. No significant difference was found for most of the inflammatory parameters, duration of hospital stay and respiratory support. Overall, patients with deficient, but not insufficient Vitamin D level had 6 times higher odds of composite poor outcome than those with normal Vitamin D (crude OR=5.18, p-value= 0.003; adjusted OR =6.3, p-value=0.043).@*Conclusion@#The inverse relationship between Vitamin D level and poor composite outcome observed in our study suggests that low Vitamin D may be a risk factor for poor prognosis among patients admitted for COVID-19.
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Diabetes Mellitus, Type 2 , Non-alcoholic Fatty Liver DiseaseABSTRACT
Inorganic arsenic (iAs) is a common carcinogen that exists in the environment. Liver, as the main target organ of arsenic metabolism, long-term exposure to iAs can ultimately lead to carcinogenesis through two stages: liver fibrosis and cirrhosis. Ferroptosis is a type of programmed cell death caused by the accumulation of iron dependent lipid peroxides that affects the normal function of mitochondria. It has been found that ferroptosis occurs during liver fibrosis. Liver fibrosis caused by iAs has been a global health problem for a long time, but so far there is no effective treatment. The discovery of ferroptosis provides a new way to solve this problem. Therefore, this article will review the research progress of the mechanism of liver injury caused by iAs and ferroptosis.
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Objective:To observe the expression levels of Toll-like receptor 4 (TLR4) signaling pathway-related proteins and their phosphorylation in the liver tissues of rats with inorganic arsenic poisoning, and to explore the role of TLR4-mediated inflammatory signaling pathway in arsenic-induced liver fibrosis injury.Methods:Eighteen healthy weanling SD rats were divided into 3 groups according to their body weight (80 - 100 g) using a random number table (6 rats in each group, half males and half females). The control group was given 10 ml/kg of normal saline by gavage. The sodium arsenite (NaAsO 2) exposure group was given 10 mg/kg of NaAsO 2 by gavage. The TAK-242 intervention group was given 10 mg/kg of NaAsO 2 by gavage, and 0.5 mg/kg of TAK-242 was also administered intraperitoneally to inhibit TLR4 after 12 weeks. All rats were administered 6 days a week for 36 weeks. At the end of the treatment, the liver tissues and serum of the rats in each group were collected. HE and Masson staining were used to observe the pathological and fibrotic changes of the liver tissues. Automatic biochemical analyzer was used to detect serum liver function indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Western blot was used to detect the expression changes of rat liver fibrosis protein α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), Vimentin and TLR4 signaling pathway-related proteins TLR4, nuclear factor κB (NF-κB)-p65 subunit (p65), NF-κB-p50 subunit (p50) and their phosphorylation p-p65 and p-p50 expression levels. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of inflammatory related factors interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-10. Results:HE and Masson staining results showed that compared with the control group, the NaAsO 2 exposure group showed significant inflammatory cell infiltration, hepatocyte necrosis and collagen fibrous deposition, while the TAK-242 intervention group showed improvement of the inflammatory cell infiltration and reduction of collagen fibrous deposition compared with the NaAsO 2 exposure group. The results of serum liver function indexes showed that ALT, AST and ALP in NaAsO 2 exposure group were increased compared with the control group, but the TAK-242 intervention group was significantly decreased compared with the NaAsO 2 exposure group ( P < 0.05). Western bolt results showed that in NaAsO 2 exposure group, the expression levels of fibrosis protein α-SMA, TGF-β1 and Vimentin (1.04 ± 0.19, 0.92 ± 0.14, 1.20 ± 0.21) and TLR4 signaling pathway-related proteins and their phosphorylation TLR4, p50, p-p50 and p-p65 (1.16 ± 0.21, 0.95 ± 0.16, 1.24 ± 0.23, 1.56 ± 0.25) were higher than the control group (0.44 ± 0.08, 0.42 ± 0.08, 0.72 ± 0.07, 0.69 ± 0.15, 0.71 ± 0.11, 0.46 ± 0.07, 0.54 ± 0.11, P < 0.05), and the TAK-242 intervention group (0.60 ± 0.13, 0.59 ± 0.16, 0.49 ± 0.11, 0.47 ± 0.08, 0.86 ± 0.09, 0.79 ± 0.14, 1.02 ± 0.17) were lower than the NaAsO 2 exposure group ( P < 0.05). There was no significant difference in the expression level of TLR4 signal pathway-related protein p65 among the three groups ( F = 14.29, P = 0.053). ELISA results showed that the secretion levels of IL-6 and TNF-α [(98.89 ± 4.58), (83.25 ± 4.57) ng/g] in rats liver tissues of the NaAsO 2 exposure group were higher than the control group [(27.30 ± 3.92), (27.77 ± 1.83) ng/g, P < 0.05], while the secretion level of IL-10 [(36.88 ± 3.86) ng/g] was lower than the control group [(77.96 ± 7.87) ng/g, P < 0.05]. In TAK-242 intervention group, IL-6 and TNF-α secretion levels [(44.32 ± 3.60), (36.51 ± 2.93) ng/g] were lower and IL-10 secretion level [(60.40 ± 4.94) ng/g] was higher compared with the NaAsO 2 exposure group ( P < 0.05). Conclusion:TLR4-mediated inflammatory signaling pathway-related proteins and their phosphorylation are highly expressed in the liver tissues of rats with inorganic arsenic poisoning, and inhibition of TLR4 signaling pathway could significantly reduce the degree of liver fibrosis injury caused by inorganic arsenic in rats.
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Objective:To analyze DNA methylation sites related to fibrosis and autophagy in human hepatic stellate cells (LX-2 cells) induced by sodium arsenite (NaAsO 2), and to screen specific methylation genes related to fibrosis and autophagy. Methods:Genome-wide DNA detection was performed using Illumina Infinium Methylation EPIC BeadChips (850K methylation chip) to derive differential methylation sites in LX-2 cells (control group) and the fibrosis and autophagy models of LX-2 cells induced by NaAsO 2(low, medium and high dose groups: the final concentrations were 5, 10, 15 μmol/L NaAsO 2, respectively, after 48 h intervention). Gene ontology (GO) function enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analysis were used to explore gene function. Results:The model of cell fibrosis and autophagy was established successfully in high dose group. The results of 850K methylation chip detection showed that there were 25 817 significant different methylation sites between the high dose group and the control group, including 12 083 hypermethylation sites and 13 734 hypomethylation sites. GO function enrichment analysis showed that the molecular functions of differentially methylated genes mainly included protein binding, ion binding, catalytic activity, enzyme binding. KEGG signaling pathway enrichment analysis showed that the pathways involved in differentially methylated genes mainly included metabolic pathway, cancer pathway, phosphatidylinositol-3-kinase-protein kinase B (PI3K-Akt) signaling pathway, endocytosis, and mitogen activated protein kinase (MAPK) signaling pathway. In the promoter region, 11 and 29 differentially methylated genes related to fibrosis and autophagy were screened, respectively.Conclusions:A large number of differential methylation sites exist in the process of NaAsO 2 induced fibrosis and autophagy of LX-2 cells. Specific methylation genes related to fibrosis and autophagy are screened out.
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@#Abstract: Objective To investigate the expression level and clinical significance of serum liver fibrosis-associated lncRNA1 (lnc-LFAR1) in patients with chronic hepatitis B cirrhosis, aiming to analyze its correlation with interleukin-6 (IL-6), interleukin-1β (IL-1β), and liver function. Methods Patients with chronic hepatitis B (CHB) cirrhosis and CHB diagnosed and treated in Dongguan City People's Hospital from March 2016 to December 2019 were selected and divided into the liver cirrhosis group (n=80) and the CHB group (n=80), and 80 healthy people with physical examination during the same period were selected as healthy group. The serum levels of lnc-LFAR1, interleukin-6 (IL-6), albumin (ALB), interleukin-1β (IL-1β) and liver function indicators, including albumin (ALB) and alanine aminotransferase (ALT) were measured and analyzed. The correlation between serum lnc-LFAR1 expression level and IL-6, IL-1β was assessed, and the levels of lnc-LFAR1, IL-6, IL-1β, ALB and ALT were compared among patients with CHB cirrhosis of different Child-Pugh grades. Results The serum levels of lnc-LFAR1, IL-6, IL-1β and ALT in the patients with liver cirrhosis [(1.85± 0.62), (41.76±13.92) ng/mL, (7.78±1.95) pg/mL, (148.37±29.67) U/L] were higher than those in the CHB group [(1.42±0.47), (23.56± 7.85) ng/mL, (5.42±1.41) pg/mL, (87.59±17.52) U/L] and the healthy group [(1.01±0.34), (6.70±2.23) ng/mL, (3.13± 0.78) pg/mL, (15.44±3.10) U/L] (P<0.05), while the ALB levels (30.54±3.82) g/L were lower than those in the CHB group (37.27±4.34) g/L and the healthy group (45.26±5.66) g/L (P<0.05). Serum lnc-LFAR1, IL-6, IL-1β and ALT levels in the CHB group were higher than those in the healthy group (P<0.05), and ALB levels were lower than those in the healthy group (P<0.05); the serum levels of lnc-LFAR1, IL-6, IL-1β in patients with CHB cirrhosis were negatively correlated with ALB (P<0.05), and positively correlated with ALT (P<0.05); the serum expression level of lnc-LFAR1 in patients with CHB cirrhosis was positively correlated with IL-6 and IL-1β (r=0.598, 0.571, P<0.05); with the increase of Child-Pugh grade, the serum levels of lnc-LFAR1, IL-6, IL-1β, and ALT in patients with CHB cirrhosis gradually increased (P<0.05), and the level of ALB gradually decreased (P<0.05). Conclusions Serum lnc-LFAR1 expression level is higher in patients with CHB cirrhosis, which is obviously related to IL-6, IL-1β, ALB and ALT. Therefore, the evaluation of serum lnc-LFAR1 expression level is helpful in the clinical assessment of the condition of CHB cirrhosis patients.
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Liver fibrosis is a compensatory response in the process of tissue repair after chronic liver injury, and it is also a necessary pathological process in the progression of a variety of chronic liver diseases. In the pathological state, the imbalance between hepatic oxidative system and antioxidant system can lead to the excessive production or insufficient clearance of reactive oxygen species (ROS)/reactive nitrogen species (RNS), which may induce the injury of hepatocytes, expand inflammatory response, and promote the development and progression of liver fibrosis. As a master regulator of oxidative stress and inflammatory response, NF-κB plays a key role in the process of liver fibrosis. Therefore, the cascade interaction between ROS/RNS and the NF-κB signaling pathway plays a guiding role in further clarifying the pathogenesis of liver fibrosis and exploring effective prevention and treatment strategies. This article reviews and discusses the interaction between ROS/RNS and the NF-κB signaling pathway and its important role in the progression of liver fibrosis, so as to provide strategies and references for targeted therapy for liver fibrosis.
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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 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.
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Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl4+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl4+PHx+MSC-EV group was higher than that in the CCl4+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.
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Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), is a common hepatic manifestation of metabolic syndrome. However, there are no effective therapy to treat this devastating disease. Accumulating evidence suggests that the generation of elastin-derived peptides (EDPs) and the inhibition of adiponectin receptors (AdipoR)1/2 plays essential roles in hepatic lipid metabolism and liver fibrosis. We recently reported that the AdipoR1/2 dual agonist JT003 significantly degraded the extracellular matrix (ECM) and ameliorated liver fibrosis. However, the degradation of the ECM lead to the generation of EDPs, which could further alter liver homeostasis negatively. Thus, in this study, we successfully combined AdipoR1/2 agonist JT003 with V14, which acted as an inhibitor of EDPs-EBP interaction to overcome the defect of ECM degradation. We found that combination of JT003 and V14 possessed excellent synergistic benefits on ameliorating NASH and liver fibrosis than either alone since they compensate the shortage of each other. These effects are induced by the enhancement of the mitochondrial antioxidant capacity, mitophagy, and mitochondrial biogenesis via AMPK pathway. Furthermore, specific suppression of AMPK could block the effects of the combination of JT003 and V14 on reduced oxidative stress, increased mitophagy and mitochondrial biogenesis. These positive results suggested that this administration of combination of AdipoR1/2 dual agonist and inhibitor of EDPs-EBP interaction can be recommended alternatively for an effective and promising therapeutic strategy for the treatment of NAFLD and NASH related fibrosis.
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ObjectiveThis study aimed to investigate the effects of eugenol on inhibiting the inflammatory activation of human umbilical cord mesenchymal stem cells （HUC-MSCs） and the pro-inflammatory phenotype of hepatic stellate cells （HSCs） in liver fibrosis， and to explore their underlying mechanisms. MethodsHUC-MSCs were cultured and identified in vitro， and the toxicity of eugenol to HUC-MSCs was evaluated by MTT method. The effect of eugenol on the migration ability of HUC-MSCs was investigated by in vitro scratch test. The expression of α-SMA， COL1A1， Smad2/3 and p-Smad2/3 of LX-2 cells activated by TGF-β1 treated with EU-MSCs-CM and MSCs-CM were detected by WB assay. EU-MSCs-CM and MSCs-CM treated THP-1 macrophages stimulated with Lipopolysaccharide （LPS） were analyzed for the expression of surface markers CD11b， CD86， and CD206 by flow cytometry. Additionally， the expression of pro-inflammatory genes TNF-α， IL-1β， and IL-6 in THP-1 macrophages was detected by qPCR. ResultsThe results of MTT method showed that the viability of the cells remained above 90% after 24 h and 48 h treatment at 0， 7.5， 15 μg/mL. In vitro scratches showed that eugenol treatment enhanced HUC-MSCs migration. WB results showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment significantly inhibited the expression of α-SMA， COL1A1， Smad2/3， and p-Smad2/3 of activated HSCs. Flow cytometry showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment had a more significant inhibitory effect on CD86， a M1-type polarization marker in THP-1 macrophages. The results of qPCR experiment showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment more significantly inhibited the expressions of TNF-α， IL-1β and IL-6 of THP-1 macrophage proinflammatory genes. ConclusionsEugenol enhances the inhibitory effect of HUC-MSCs on inflammatory activation of HSCs， possibly by regulating TGF-β1/Smads signaling pathway. It also enhances the inhibitory effect of HUC-MSCs on the pro-inflammatory phenotype of macrophages. Proinflammatory macrophages can promote inflammatory activation of HSCs.
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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.
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Humans , Hepatic Stellate Cells , Lipopolysaccharides/pharmacology , Collagen Type I , Exosomes , Macrophages , MicroRNAsABSTRACT
Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.
Subject(s)
Mice , Animals , Interferon-gamma/metabolism , Interleukin-17/metabolism , Interleukin-33/metabolism , Cytokines/metabolism , Carbon Tetrachloride , Th2 Cells , Interleukin-4/metabolism , Liver Cirrhosis/pathology , Th1 Cells , Th17 Cells/pathology , ImmunityABSTRACT
Based on network pharmacology, molecular docking, and in vitro experimental verification, this study aims to explore the effect of Albiziae Cortex-Tribuli Fructus combination on HSC-LX2 pyroptosis. Specifically, the targets of Albiziae Cortex, Tribuli Fructus, and hepatic fibrosis were retrieved from an online database and CNKI, and "drug-component-target" network and "drug-component-target-disease" network were constructed. Protein-protein interaction(PPI) network was established based on STRING. Metascape was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and the mechanism of Albiziae Cortex-Tribuli Fructus combination against liver fibrosis was predicted. Molecular docking was used to verify some of the results of network pharmacology, and in vitro experiment was carried out to further verify the above conclusions. According to the results of network pharmacological analysis, 25 active components and 439 targets of Albiziae Cortex-Tribuli Fructus combination and 152 anti-liver fibrosis targets were screened out, including nucleotide-binding oligomerization domain and leucine-rich-repeat-and pyrin-domain-containing 3(NLRP3) and caspase-1. The key targets were involved in 194 KEGG pathways in which the NOD-like receptor signaling pathway topped. The binding common targets were related to pyroptosis. The results of in vitro experiment showed that the pair-containing serum reduced the proliferation rate of HSC-LX2 and the content of reactive oxygen species(ROS), interleukin-18(IL-18), and interleukin-1β(IL-1β)(P<0.05). Western blot and qRT-PCR suggested that the protein and gene expression of NLRP3, caspase-1, α-smooth muscle actin(α-SMA), and gasdermin D(GSDMD) in HSC-LX2 increased after AngⅡ stimulation, and the expression decreased after the intervention of pair-containing serum(P<0.05). In summary, the pair-containing serum can inhibit the classic pathway of pyroptosis, which may be the anti-liver fibrosis mechanism. This is consistent with the predicted results of network pharmacology.
Subject(s)
Humans , Hepatic Stellate Cells , Network Pharmacology , Molecular Docking Simulation , NLR Family, Pyrin Domain-Containing 3 Protein , Caspase 1/genetics , Fibrosis , Drugs, Chinese Herbal/pharmacologyABSTRACT
As a natural compound with high efficiency and low toxicity, cryptotanshinone (CTS) has a good anti-fibrosis effect in various organs and tissues. However, its mechanism of action has not been clearly defined, and there is no systematic literature review to describe its potential anti-fibrosis mechanism. The efficacy and mechanism of cryptotanshinone in the treatment of fibrosis in various organs were summarized and the use prospects were put forward in this paper.
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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.