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OBJECTIVE To investigate the intervention effect and potential mechanism of breviscapine on hepatic fibrosis (HF) in rats based on the transforming growth factor-β(1 TGF-β1)/Smad2/extracellular signal-regulated protein kinase 1(ERK1) and Kelch-like epichlorohydrin-associated protein 1(Keap1)/nuclear factor-erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathways. METHODS Totally 60 rats were randomly divided into normal control group, model group, breviscapine low-dose, medium-dose and high-dose groups (5.4, 10.8, 21.6 mg/kg), and colchicine group (positive control, 0.45 mg/kg), with 10 rats in each group, half male and half female. Except for the normal control group, HF model of the other groups was induced by carbon tetrachloride. Subsequently, each drug group was given corresponding medicine by gavage once a day for 28 days. The liver appearance of rats in each group was observed and their liver coefficients were calculated. The levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST)in serum, those of ALT, AST, superoxide dismutase (SOD),malondialdehyde (MDA) and glutathione peroxidase (GSH- Px) in liver tissue were detected. The liver tissue inflammatory and fibrotic changes were observed. The protein and mRNA expressions of TGF-β1, Smad2, ERK1, Nrf2, Keap1 and HO-in liver tissue were detected. RESULTS Compared with the normal control group, the model group showed large areas of white nodular lesions in the liver, obvious inflammatory cell infiltration and collagen fiber deposition. The body weight, the levels of SOD and GSH-Px in liver tissue, the protein and mRNA expressions of Nrf2 and HO-1 were significantly lowered in the model group (P<0.05); the liver coefficient, the percentage of Masson staining positive area, ALT and AST levels of serum and liver tissue, MDA level of liver tissue, the protein and mRNA expressions of TGF-β1, Smad2, ERK1 and Keap1 were significantly increased (P<0.05). Compared with the model group, the liver lesions of rats in each drug group were improved, and the above quantitative indexes were generally reversed (P<0.05). CONCLUSIONS Breviscapine has a good intervention effect on HF rats, which may be related to inhibiting TGF-β1/Smad2/ERK1 pathway for anti-fibrosis and regulating Keap1/Nrf2/HO-1 pathway to inhibit oxidative stress.
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OBJECTIVE To investigate the ameliorative effects and mechanism of Sanwei ganlu on hepatic fibrosis in rats. METHODS The rats were randomly divided into normal group, model group, silibinin group (positive control, 50 mg/kg), and Sanwei ganlu low-dose, medium-dose, and high-dose groups (80, 250, 800 mg/kg). Except for normal group, hepatic fibrosis rat models were established by intraperitoneal injection of CCl4 in the other groups of rats. Starting from the 6th week of modeling administration, they were given normal saline or corresponding drugs intragastrically at the same time. At the end of the ninth-week experiment, liver and spleen indexes of rats were calculated; the pathological structure and fibrosis changes of liver tissue were observed by HE, Masson and Sirus Red staining. The contents of alanine transaminase (ALT), aspartate transaminase (AST), procollagen type Ⅲ (PC Ⅲ), collagen type Ⅳ (COL-Ⅳ), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β in serum, and hyaluronic acid (HA) and laminin (LN) in liver tissue were all detected. RESULTS Compared with the model group, the liver injury and collagen fiber deposition of rats were improved to different extents in Sanwei ganlu groups and silibinin group; the contents of ALT, AST, PC Ⅲ, COL-Ⅳ, IL-6, TNF-α and IL-1β in serum as well as the contents of HA and LN in liver tissue significantly decreased (P<0.05 or P<0.01). CONCLUSIONS Sanwei ganlu can alleviate the progression of hepatic fibrosis in rats, possibly by inhibiting the synthesis of collagen fiber, reducing transaminase content, down-regulating the levels of HA, LN, PC Ⅲ and COL-Ⅳ, and reducing the inflammatory response.
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ObjectiveTo investigate the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) in the treatment of mice with liver fibrosis and its mechanism. MethodsA total of 18 specific pathogen-free C57BL/6 mice, aged 6 weeks, were selected and divided into control group (n=6), carbon tetrachloride (CCl4) model group (CCl4 group, n=6), and hUCMSCs treatment group (MSC group, n=6) using a random number table. The mice in the CCl4 group and the MSC group were given intraperitoneal injection of CCl4 solution to establish a mouse model of liver fibrosis, while those in the control group were injected with the same dose of corn oil, and the mice in the MSC group were injected with hUCMSCs via the caudal vein during the injection of CCl4. At the end of week 8, mouse serum was collected, and the mice were sacrificed to collect and fix the liver. Enzyme-linked immunosorbent assay was used to measure the levels of inflammatory factors; an automatic biochemical detector was used to measure liver function parameters; HE staining, Masson staining, Sirius Red staining, and α-SMA immunofluorescence assay were used to evaluate liver fibrosis. Hepatic stellate cells (HSCs) stimulated by TGF-β were co-cultured with hUCMSCs in the medium with or without chitinase-3 like-protein-1 (CHI3L1), and Western blot was used to measure the expression levels of proteins. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett’s t-test was used for further comparison between two groups. ResultsMasson staining and Sirius Red staining showed that the CCl4 group had a significantly higher degree of fibrosis than the control group (both P<0.05), and the MSC group had significant alleviation of fibrosis compared with the CCl4 group (both P<0.05). Compared with the control group, the CCl4 group had significant increases in the levels of interleukin-1β, interleukin-6 (IL-6), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) (all P<0.05), and compared with the CCl4 group, the MSC group had significant reductions in the levels of IL-6, AST, ALT, and ALP (all P<0.05). The CCl4 group had significantly higher expression levels of CHI3L1 and α-SMA than the control group and the MSC group (all P<0.05). The cell culture experiment showed that the MSC+HSC group had a significantly higher expression level of Bax than the HSC group and the MSC+CHI3L1 group (both P<0.05), suggesting that CHI3L1 reversed the pro-apoptotic effect of MSC on activated HSCs. ConclusionThis study shows that hUCMSCs can improve liver fibrosis in mice, possibly by inhibiting CHI3L1 to promote the apoptosis of HSCs.
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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.
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Objective To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Methods Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson’s trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Results Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson’s trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). Conclusions E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.
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Objective To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. Methods C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. Results Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = −3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = −0.723, P > 0.05), TNF-α (t = −0.659, P > 0.05), IL-4 (t = −0.263, P > 0.05), IL-10 (t = −0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = −4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = −1.902, P > 0.05), IL-4 (t = −1.333, P > 0.05), IL-10 (t = −1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). Conclusions During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.
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Aim To investigate the effects of 2-dode-cyl-6-methoxycyclohexa-2 , 5-diene-l, 4-dione ( DM-DD) on resisting hepatic fibrosis induced by carbon tetrachloride ( CC14 ) in rats and the underlying mechanisms , with a specific focus on the TGF-pi/Smads signaling pathway. Methods The hepatic fibrosis model was replicated using 50% CC14. Various parameters, including levels of aspartate transferase ( AST) , ala-nine transferase ( ALT ) , albumin/globulin ( A/G ) , total protein (TP) , total bilirubin (T-BIL) , hyaluron-ic acid ( HA ) , laminin ( LN ) , collagen type Ж ( Col Ж) , and collagen type IV(ColIV) in the blood, were measured. Liver tissue lesions and fiber formation were observed using HE and Masson staining. The expression levels of a smooth muscle actin (a-SMA) , collagen type I ( Col I ) , transformed growth factor (TGF-pi), Smad2, and Smad7 proteins were assessed using immunohistochemistry. a-SMA, Coll, TGF-pi, and Smad7 mRNA levels in liver tissue were measured by RT-PCR. Additionally, the expression levels of TGF-pi, Smad4, and Smad7 proteins in liver tissue were determined by Western blot. Results In comparison to the normal control group, the model group exhibited significantly elevated levels of AST, ALT, TP, T-BIL, HA, LN, Col Ш and Col IV in serum. But A/G level notably decreased. Successful modeling was confirmed by the presence of extensive fiber formations observed through HE and Massonstaining in liver tissue. The DMDD administration group demonstrated a notable decrease levels of AST, ALT, TP, T-BIL, HA, LN, Col III, and CollV, but A/G was significantly elevated when compared to the model group. Furthermore, a-SMA, Coll, TGF-f31, Smad2 and Smad4 mRNA and protein levels in the DMDD administration group were significantly reduced, while Smad7 significantly declined. HE and Masson staining results reflected a marked reduction in fibrous hyper-plasia. Conclusion DMDD exhibits a protective effect against CCl4-induced hepatic fibrosis, and its mechanism appears to be associated with the TGF-fJl/ Smads signaling pathway.
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OBJECTIVE To explore the effect and mechanism of the alcoholic extract from Scabiosa comosa against hepatic fibrosis (HF). METHODS Intragastrical administration of carbon tetrachloride was given to induce HF model. By observing the pathological changes in liver tissue, mRNA and protein expressions of HF indexes [α-smooth muscle actin (α-SMA), collagen type Ⅰ] and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway-related factors were detected, and the improvement effects and possible mechanism of low-dose, medium-dose and high-dose (50, 100, 200 mg/kg) of alcoholic extract from S. comosa on HF model rats were investigated. Drug-containing serum was prepared by intragastrical administration of alcoholic extract from S. comosa at a concentration of 1 800 mg/(kg·d) (calculated by the amount of raw material). The effects of drug- containing serum of alcoholic extract from S. comosa on the expression of miRNA-21 were observed through the intervention of HSC-T6 cells with low, medium and high concentrations of drug-containing serum of alcoholic extract from S. comosa (diluted to 10%, 15%, 20%). miRNA-21 mimics or inhibitors were used to transfect HSC-T6 cells, and the mRNA and protein expressions of factors related to the PI3K/Akt signaling pathway were detected. RESULTS The results of in vivo experiments showed that low, medium and high doses of alcoholic extract from S. comosa significantly ameliorated the histopathological changes in liver tissue of HF rats, and the percentage of collagen was significantly reduced (P<0.01); mRNA and protein expressions of the indicators related to HF as well as PI3K and Akt were significantly reduced (P<0.01), and mRNA and protein expressions of phosphatase and tensin homolog deleted on chromosome ten (PTEN) were increased in liver tissue of rats (P<0.01). The results of in vitro experiments showed that drug-containing serum of alcoholic extract from S. comosa significantly inhibited the expression of miRNA-21 at low, medium and high concentrations (P<0.01); whereas after transfection with miRNA-21 mimics, it was found that miRNA-21 mimics significantly increased mRNA and protein expressions of PI3K and Akt (P<0.01), while significantly decreased mRNA and protein expressions of PTEN (P<0.01); after transfection with miRNA-21 inhibitor, the changes of above indexes were opposite to the above results (P<0.01). CONCLUSIONS Alcoholic extracts of S. comosa may inhibit the PI3K/Akt signaling pathway by affecting the expression of miRNA-21, so as to achieve the effect of anti-hepatic fibrosis.
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ObjectiveTo investigate the effect of Dendrobium officinale polysaccharide (DOP)on CCl4-induced hepatic fibrosis(HF)and its mechanism. MethodsA total of 56 male SD rats were randomly divided into seven groups: normal group(NG),model group(MG),colchicine group(CG, 0.1 mg/kg), Fuzheng Huayu group(FG, 0.45 g/kg),low-dose DOP group(LDG, 0.05 g/kg),middle-dose DOP group(MDG, 0.1 g/kg)and high-dose DOP group(HDG,0.2 g/kg),with 8 rats in each group. HF rat model was established by subcutaneous injection with 40% CCl4 olive oil mixture, every 3-day for 10 weeks. At the end of the sixth week, the drug groups were treated with colchicine, Fuzheng Huayu and DOP solution by gavage respectively, once a day for 4 weeks. NG and MG groups were similarly handled with an equal amount of 0.9 % normal saline. Liver histopathology was detected using hematoxylin-eosin (HE), Masson and Sirius red staining; blood biochemistry was tested for liver function and four indicators of HF; RT-qPCR and Western Blot were used to measure the expression of α-SMA, Col-I, E-cadherin, and ZEB1 genes and proteins in the liver tissues of rats, respectively. ResultsHE, Masson, and Sirius red staining showed that the liver tissue of MG rats had typical pathologic features of HF, and the degree of HF was alleviated in LDG, MDG, and HDG rats, respectively. Liver function test results showed that the serum AST, TBIL, and AKP levels were significantly lower in LDG, MDG, and HDG, compared with those of the MG (P < 0.05 or < 0.01). Meanwhile, ALT levels in serum deceased remarkably except in LDG (P < 0.05 or < 0.01). The four results of HF showed that the serum HA, LN, PC-Ⅲ, and COL-Ⅳ levels in LDG, MDG, and HDG rats were significantly decreased compared with those of the MG (P < 0.05 or < 0.01). The relative expressions of α-SMA, COL-I, and ZEB1 genes and proteins were significantly decreased in the liver tissues of LDG, MDG, and HDG (P < 0.05 or < 0.01), and the relative expression of E-cadherin gene and protein increased (P < 0.05 or < 0.01). In addition, the expressions of HA, α-SMA, COL-I, ZEB1 and E-cadherin were dependent on the dose of DOP. ConclusionDOP alleviated the degree of CCl4 induced HF in rats by inhibiting the epithelial-mesenchymal transition in liver tissue.
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ObjectiveTo investigate the preventive and therapeutic effects of Tiaogan Huaxian pills combined with entecavir on hepatic fibrosis in chronic hepatitis B with liver Qi stagnation, spleen deficiency, and blood stasis syndrome and its effect on diffusion-weighted imaging (DWI). MethodClinical data of 117 patients with liver disease who visited the Department of Hepatology at the First Affiliated Hospital of Guangxi University of Chinese Medicine from January 2021 to April 2022 were retrospectively analyzed. According to different treatment plans, they were divided into a control group (59 cases) and a treatment group (58 cases). Both groups of patients received entecavir-based etiology treatment, and the treatment group added Tiaogan Huaxian pills on the basis of basic treatment. Both groups were treated for 24 weeks. Before and after treatment, the two groups were compared in terms of alanine aminotransferase (ALT), advanced surgical technologies (AST), total bilirubin (TBil), hepatitis B virus (HBV)-DNA conversion rate, liver stiffness measurement (LSM), four items of liver fibrosis (hyaluronidase, type Ⅲ pro-collagen, type Ⅳ collagen, and laminin), the fibrosis index based on four factors (FIB-4), the aspartate aminotransferase to platelet ratio index (APRI), the apparent diffusion coefficient (ADC) value in magnetic resonance imaging (MRI), and traditional Chinese medicine symptom scores, so as to analyze the efficacy of the two groups. ResultBefore treatment, there was no significant difference in ALT, AST, TBil, LSM, four items of liver fibrosis, FIB-4, APRI, HBV-DNA conversion rate, ADC value, and traditional Chinese medicine symptom scores between the two groups. After treatment, both groups of patients showed significant reductions in ALT, AST, TBil, LSM, hyaluronidase, type Ⅲ pro-collagen, type Ⅳ collagen, laminin, FIB-4, and APRI (P<0.05) and a significant increase in ADC value (P<0.05) and HBV-DNA conversion rate (P<0.01). The traditional Chinese medicine symptom score of the treatment group decreased significantly (P<0.05). Compared with the control group after treatment, the effective rate of clinical traditional Chinese medicine in the treatment group was 91.38% (53/58), which was significantly higher than that of the control group (54.23%, 32/59) (Z=-4.325, P<0.01). In the treatment group, ALT, AST, TBil, LSM, hyaluronidase, type Ⅲ pro-collagen, type Ⅳ collagen, laminin, FIB-4, APRI, and traditional Chinese medicine symptom scores all decreased significantly (P<0.05), and the increase in ADC values was more significant (P<0.05), while the difference in HBV-DNA conversion rate was not statistically significant. There were no serious adverse reactions or events in either group. ConclusionTiaogan Huaxian pills combined with entecavir have significant clinical efficacy in the treatment of hepatic fibrosis in chronic hepatitis B, which can reduce liver inflammation activity, delay hepatic fibrosis progression, and reduce traditional Chinese medicine symptom scores. It is worthy of clinical promotion and application.
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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.
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ObjectiveTo investigate the effect of phytoestrogen biochanin A (BCA) on liver fibrosis induced by CCl4 in female mice with bilateral oophorectomy (ovariectomized) and its mechanism. MethodsA total of 50 ovariectomized Kunming mice were selected and given intraperitoneal injection of CCl4 to establish a model of liver fibrosis, and then according to body weight, they were randomly divided into model group, positive control group, and low-, middle-, and high-dose BCA groups, with 10 mice in each group. In addition, 10 female mice in the same litter were given resection of a small amount of adipose tissue near both ovaries to establish the sham-operation group. The mice in the positive control group were given estradiol 2 mg/kg by gavage, and those in the low-, middle-, and high-dose BCA groups were given BCA by gavage at a dose of 25, 50, and 100 mg/kg, respectively, once a day for 7 consecutive weeks; the mice in the sham-operation group and the model group were given an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage. The mice were anesthetized and sacrificed after administration to collect samples. Liver index and uterus index were measured; HE staining and Masson staining were used to observe liver histopathological changes; the biochemical analysis was used to measure the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); ELISA was used to measure the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in liver tissue, and Western blot was used to measure the relative protein expression levels of collagen Ⅰ, transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), estrogen receptor beta (ERβ), and p-NF-κBp65/NF-κBp65 in liver tissue. The t-test was used for comparison of continuous data between two groups; a one-way analysis of various was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and further comparison between two groups. ResultsCompared with the sham-operated group, the model group had a significant increase in liver index and a significant reduction in uterus index, as well as significant increases in the activities of serum AST and ALT, the levels of IL-6 and TNF-α in liver tissue, and the protein expression levels of collagen Ⅰ, TGF-β1, α-SMA, and p-NF-κBp65/NF-κBp65 in liver tissue (all P<0.05), with no significant change in the expression of ERβ in liver tissue (P>0.05), and the model group showed significant fibrosis lesions in the liver, such as hepatocyte edema, steatosis, and necrosis with inflammatory cell infiltration and hyperplasia, deposition, and staggered distribution of collagen fibers. Compared with the model group, the low-, middle-, and high-dose BCA groups had significant reductions in liver index, the activities of serum AST and ALT, the levels of IL-6 and TNF-α, and the protein expression levels of collagen Ⅰ, TGF-β1, α-SMA, and p-NF-κBp65/NF-κBp65 in liver tissue (all P<0.05), with no significant change in uterine index (P>0.05), as well as a significant increase in the protein expression level of ERβ in liver tissue (P<0.05) and varying degrees of improvement in liver fibrosis lesions. ConclusionBCA can effectively improve CCL4-induced liver fibrosis in ovariectomized female mice, possibly by upregulating ERβ to inhibit the NF-κB signaling pathway and then alleviating inflammatory response.
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Hepatic fibrosis (HF) is a pathological process of abnormal repair of liver tissue structure caused by chronic liver injury, and its pathogenesis has not been fully clarified. Related studies have shown that programmed cell death may be associated with the onset of HF, and traditional Chinese medicine (TCM) has a significant effect in regulating programmed cell death to intervene against HF. This article reviews the main mechanism of the influence of programmed cell death on HF and discusses the possible mechanism of TCM regulation of programmed cell death in improving HF, which provides new ideas for TCM prevention and treatment of HF.
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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.
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ObjectiveTo investigate the value of biliary score and hepatic signal intensity-to-muscle signal intensity ratio (HMR) obtained by multiphase contrast-enhanced MRI scan using Gd-EOB-DTPA in evaluating the pathological grade of liver fibrosis. MethodsA retrospective analysis was performed for the MRI and clinical data of 51 patients with chronic hepatitis B liver fibrosis in Wuming Hospital Affiliated to Guangxi Medical University from January 2020 to May 2023. The 51 patients with liver fibrosis were divided into low-grade group (S1-S2) and high-grade group (S3-S4). GE Architact 3.0T MR scanner was used to perform MRI scans, including routine plain scan and contrast-enhanced scan at arterial phase, portal venous phase, delayed phase, hepatobiliary phase, and excretory phase, and biliary score and HMR were measured for the patients with different grades of liver fibrosis. The t-test was used for comparison of continuous data between groups, and the chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups. The receiver operating characteristic (ROC) curve was plotted to evaluate the value of MRI indicators in determining the pathological grade of liver fibrosis. ResultsAmong the 51 patients with liver fibrosis, there were 30 patients in the low-grade group and 21 in the high-grade group. Compared with the high-grade group, the low-grade group had significantly higher biliary score (3.67±0.55 vs 2.57±0.75, t=6.05, P<0.001) and HMR at portal venous phase (2.38±0.76 vs 1.97±0.18, t=2.41, P=0.020), delayed phase (2.48±0.70 vs 1.99±0.27, t=3.09, P=0.003), and hepatobiliary phase (4.10±0.63 vs 3.16±0.47, t=5.81, P<0.001). The above indicators had an area under the ROC curve (AUC) of 0.86, 0.79, 0.82, and 0.88, respectively, in distinguishing low- and high-grade liver fibrosis, with a positive rate of 70%, 63.3%, 83.3%, and 96.7%, respectively, and a negative rate of 90%, 95.2%, 74.1%, and 100%, respectively, in the diagnosis of high-grade liver fibrosis. Biliary score combined HMR had an AUC of 0.95, with a positive rate of 85.7% and a negative rate of 96.7%. ConclusionBiliary score and HMR at hepatobiliary phase obtained by multiphase contrast-enhanced MRI scan using Gd-EOB-DTPA has a relatively high diagnostic efficacy in distinguishing between low- and high-grade liver fibrosis and a certain guiding value for the diagnosis and treatment of liver fibrosis in clinical practice.
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ABSTRACT Objective: To investigate nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and hepatic fibrosis in biopsies of people with obesity who underwent bariatric surgery and examine the possible association of different variables with a diagnosis of NAFLD and NASH. Materials and methods: Epidemiological, clinical and laboratory data from 574 individuals with obesity of both genders seen by the same physician between 2003 and 2009 who had a liver biopsy during bariatric surgery were examined. Results: Of the 437 patients included, 39.8% had some degree of liver fibrosis, 95% had a histologic diagnosis of NAFLD, and the risk factors were age ≥ 28 years and Homeostatic Model Assessment (HOMA) ≥ 2.5 (p = 0.001 and p = 0.016, respectively). In the NAFLD group, NASH was present in 26% of patients and the associated factors were aspartate aminotransferase and alanine aminotransferase index (AST/ALT) > 1, high-density lipoprotein cholesterol (HDL-c) < 40 mg/dL, total cholesterol (TC) ≥ 200 mg/dL, gamma-glutamyl transferase (GGT) > 38 U/L and triglycerides (TG) levels > 150 mg/dL. The independent risk factors were low HDL-c, elevated AST/ALT and high TG. Conclusion: The variables associated with a diagnosis of NAFLD were HOMA ≥ 2.5 and age ≥ 28 years. NASH was associated with low HDL-c, high TG and AST/ALT ≤ 1.
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Objective To screen differentially expressed long non-coding RNAs (lncRNAs) in the liver of mice infected with Schistosoma japonicum during the chronic pathogenic stage and identify their functions, so as to provide insights into unravelling the role of lncRNAs in S. japonicum infection-induced liver disorders. Methods Twenty 6-week-old C57BL/6 mice were randomly divided into two groups, of 10 animals each group. Each mouse in the experimental group was infected with (15 ± 2) S. japonicum cercariae via the abdomen for modeling chronic S. japonicum infection in mice, and distilled water served as controls. All mice were sacrificed 70 days post-infection, and mouse liver specimens were sampled for RNA extraction and library construction. All libraries were sequenced on the Illumina NovaSeq 6000 sequencing platform. Data cleaning was performed using the fastp software, and reference genome alignment and gene expression (FPKM) calculation were performed using the HISAT2 software. Potential lncRNA sequences were predicted using the software CNIC, CPC, Pfam, and PLEK, and potential lncRNAs were screened. Differentially expressed lncRNAs were screened with the DESeq2 software and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to identify biological processes and metabolic pathways involved in target genes of differentially expressed lncRNAs. Results A total of 333 potential lncRNAs were screened, and 67 were identified as differentially expressed lncRNAs, including 49 up-regulated and 18 down-regulated lncRNAs. A total of 53 target genes were predicted for differentially expressed lncRNAs. GO enrichment analysis showed that these target genes were mainly enriched in biological process and molecular function, among which Sema7a, Arrb1, and Ccl21b genes may be hub target genes for positive regulation of extracellular regulated protein kinase 1 (ERK1) and ERK2 cascades and may participate in the regulation of collagen expression. KEGG enrichment analysis showed that the target genes of differentially expressed lncRNAs were mainly enriched in cytokine-cytokine receptor interaction, viral protein interactions with cytokines and cytokine receptors, chemokine signaling pathway, and nuclear factor kappa-B (NF-κB) signaling pathway. Conclusions This study identifies differentially expressed lncRNAs and functional enrichment of their target genes in the liver of mice during the chronic pathogenic stage of S. japonicum infection. Up-regulated lncRNAs may affect biological processes of ERK1/2 cascades and chemokine signaling pathways via target genes Sema7a, Arrb1, and Ccl21b, thereby affecting collagen expression and inflammatory signal pathways, ultimately affecting the development of liver disorders.
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ObjectiveTo investigate the population distribution of non-alcoholic fatty liver disease before and after renaming and the association between the types of metabolic risk factors (MRF) for metabolic dysfunction-associated steatotic liver disease (MASLD) and advanced liver fibrosis. MethodsThis study was conducted among 515 patients who were admitted to The Affiliated Hospital of Xuzhou Medical University and Wuxi Fifth People’s Hospital from January 2019 to January 2022 and had hepatocyte steatosis ≥5% by liver biopsy. Among these patients, 2 patients did not meet the diagnostic criteria for nonalcoholic fatty liver disease (NAFLD) and metabolic associated fatty liver disease (MAFLD), respectively, and were classified as steatotic liver disease (SLD) with other specific causes, and the other 513 patients were divided into MASLD group with 275 patients, comorbid group with 216 patients (MASLD comorbid with other liver diseases), and cryptogenic SLD group with 22 patients. The above groups were compared in terms of clinical features, laboratory markers, and advanced liver fibrosis. The MASLD patients with different types of MRF were compared in terms of clinical features, laboratory markers, and advanced liver fibrosis, and the risk factors for advanced liver fibrosis in patients with MASLD were analyzed. The Kruskal-Wallis H test was used for comparison of continuous data with skewed distribution between multiple groups and further comparison between two groups; the chi-square test was used for comparison of categorical data between multiple groups, and Bonferroni correction was used for further comparison between two groups. The logistic regression analysis was used to identify the risk factors for liver fibrosis. ResultsAmong the 515 patients with SLD, 297 patients (57.7%) met the diagnostic criteria for NAFLD, among whom 22 were classified as cryptogenic SLD and 275 met the diagnostic criteria for MASLD, and 467 (90.7%) were diagnosed with MAFLD. There were significant differences between the three groups in sex, body mass index (BMI), gamma-glutamyl transpeptidase, triglyceride, cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, fasting plasma glucose, NAFLD fibrosis score (NFS), fibrosis-4 (FIB-4), and F3-4 (all P<0.05). Compared with the MASLD group and the cryptogenic SLD group, the comorbid group had the highest proportion of patients with advanced liver fibrosis (P<0.001). With the increase in the type of MRF, the patients tended to have an older age, a significantly higher proportion of female patients, a higher possibility of hypertension and diabetes, and higher levels of metabolic parameters including BMI, blood lipids, and blood glucose (all P<0.05). With the increase in the types of MRF in MASLD patients, they tended to have significantly higher noninvasive fibrosis scores (NFS and FIB-4) and a significantly higher proportion of patients with advanced liver fibrosis (P<0.05). The multivariate logistic regression analysis showed that age ≥50 years (odds ratio [OR]=2.622, 95% confidence interval [CI]: 1.091 — 6.300, P=0.031) and the increase in the type of MRF (OR=1.876, 95%CI: 1.194 — 2.947, P=0.006) were independent risk factors for MASLD with severe liver fibrosis. ConclusionThe new definition of MASLD is based on the positive identification of MRF, and the reclassified population of MASLD is smaller than that of MAFLD, with little difference from that of NAFLD. In addition, age ≥50 years and the increase in the type of MRF are independent risk factors for MASLD with advanced liver fibrosis.
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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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Caveolin-1 (CAV1) is a structural protein of caveolae on the plasma membrane and is an important regulatory factor for liver function. CAV1 regulates hepatic lipid deposition, lipid and glucose metabolism, mitochondrial function, and hepatocyte proliferation through various molecular pathways. Therefore, CAV1 plays a crucial role in maintaining liver physiology during the metabolic regulatory processes such as hepatic steatosis and hepatocyte proliferation. Furthermore, CAV1 is also involved in the development and progression of different types of liver injury, hepatitis, and liver cirrhosis. This article reviews the role of CAV1 in liver-related diseases and its mechanism in the regulation of liver macrophages, so as to provide a theoretical basis for targeting CAV1 in the treatment of liver-related diseases.