Prevalence and impact of Sjögren's syndrome in primary biliary cholangitis: a systematic review and meta-analysis.

INTRODUCTION AND OBJECTIVES: We performed a systematic review and meta-analysis to evaluate the prevalence of concomitant Sjögren's syndrome (SS) with primary biliary cholangitis (PBC) in adults and quantify the impact of SS on PBC. METHODS: PubMed, Web of Science and Cochrane library were searched using subject terms and predefined inclusion and exclusion criteria. RESULTS: Seventeen articles were included. The prevalence of SS in PBC patients ranged from 3.5 to 73% (35% pooled) (95% CI: 28-41%; p < 0.01). Seven studies included various biochemical indicators, including alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT), total bilirubin (TBiL), albumin (ALB) and platelet (PLT), and immunological indexes including IgG, IgM, antinuclear antibody (ANA), anti-mitochondrial antibody (AMA), AMA-M2 and anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies. Meta-analysis showed that there were no significant differences in ALT, AST, ALP, γ-GT, TBiL and IgM levels between PBS and PBC with SS. Pooled analysis showed that ALB (MD=0.82; 95% CI: 0.08-1.56) and PLT (MD=30.41; 95% CI: 10.16-50.66) levels were lower, IgG levels (MD=-1.55; 95% CI: -2.39 to -0.72) were higher, and the positive ratios of ANA (RR=0.92; 95% CI: 0.87-0.98), AMA (RR=0.94; 95% CI: 0.89-0.98), AMA-M2 (RR=0.77; 95% CI: 0.70-0.85) and anti-Ro/SSA antibodies (RR=0.29; 95% CI: 0.08-1.01) were significantly higher in PBC patients with SS than in PBC patients. CONCLUSIONS: Our study confirms that SS is common in PBC. Comorbid SS appears to influence the clinical phenotype of PBC and may therefore influence the management of PBC.

N-glycosylation of CREBH improves lipid metabolism and attenuates lipotoxicity in NAFLD by modulating PPARα and SCD-1.

INTRODUCTION: Cyclic adenosine monophosphate (AMP)-responsive element-binding protein H (CREBH), an endoplasmic reticulum-anchored transcription factor essential for lipid metabolism and inflammation in nonalcoholic fatty liver disease (NAFLD), is covalently modified by N-acetylglucosamine. Glycosylation is a ubiquitous type of protein involved in posttranslational modifications, and plays a critical role in various biological processes. However, the mechanism of glycosylated CREBH remains poorly understood in NAFLD. METHODS: CREBH glycosylation mutants were obtained by site-mutation methods. After transfection with plasmids, AML-12, LO2, or HepG2 cells were treated with palmitic acid (PA) proteolysis, tunicamycin (Tm), or their combination. Glycosyltransferase V (GnT-V) was used induce hyperglycosylation to further understand the effect of CREBH. In addition, glycosylation mutant mice and hyperglycosylated mice were generated by lentivirus injection to construct two kinds of NAFLD animal models. The expression of NAFLD-related factors was detected to further verify the role of N-linked glycosylation of CREBH in lipid and sterol metabolism, inflammation, and lipotoxicity. RESULTS: N-glycosylation enhanced the ability of CREBH to activate transcription and modulated the production of peroxisome proliferator-activated receptor alpha (PPARα) and stearoyl-CoA desaturase-1 (SCD-1) activity by affecting their promoter-driven transcription activity and protein interactions, leading to reduce lipid deposition and attenuate lipotoxicity. Deglycosylation of CREBH induced by Tm could inhibit the proteolysis of CREBH induced by PA. The addition of unglycosylated CREBH to cells upregulates gene and protein expression of lipogenesis, lipotoxicity, and inflammation, and aggravates liver damage by preventing glycosylation in cells, as well as in mouse models of NAFLD. Furthermore, increased N-glycosylation of CREBH, as achieved by overexpressing GnT-V could significantly improve liver lesion caused by unglycosylation of CREBH. CONCLUSION: These findings have important implications for the role of CREBH N-glycosylation in proteolytic activation, and they provide the first link between N-glycosylation of CREBH, lipid metabolism, and lipotoxicity processes in the liver by modulating PPARα and SCD-1. These results provide novel insights into the N-glycosylation of CREBH as a therapeutic target for NAFLD.

New insights into BMP9 signaling in liver diseases.

Bone morphogenetic protein 9 (BMP9) is a recently discovered cytokine mainly secreted by the liver and is a member of the transforming growth factor ß (TGF-ß) superfamily. In recent years, an increasing number of studies have shown that BMP9 is associated with liver diseases, including nonalcoholic fatty liver disease (NAFLD), liver fibrosis and hepatocellular carcinoma (HCC), and BMP9 signaling may play dual roles in liver diseases. In this review, we mainly summarized and discussed the roles and potential mechanisms of BMP9 signaling in NAFLD, liver fibrosis and HCC. Specifically, this article will provide a better understanding of BMP9 signaling and new clues for the treatment of liver diseases.

Blood biochemical characteristics of patients with coronavirus disease 2019 (COVID-19): a systemic review and meta-analysis.

Objective Recently, there have been several studies on the clinical characteristics of patients with coronavirus disease 2019 (COVID-19); however, these studies have mainly been concentrated in Wuhan, China; the sample sizes of each article were different; and the reported clinical characteristics, especially blood biochemical indices, were quite different. This study aimed to summarize the blood biochemistry characteristics of COVID-19 patients by performing a systemic review and meta-analysis of published studies. Methods Comprehensive studies were screened from PubMed, Embase, and Cochrane Library through March 11, 2020. The inclusion criteria included studies investigating the biochemical indexes of patients with COVID-19. The statistical software R3.6.3 was used for meta-analysis. Results Ten studies including 1745 COVID-19 patients met the inclusion criteria for our meta-analysis. Meta-analysis showed that 16% and 20% of patients with COVID-19 had alanine transaminase (ALT) and aspartate aminotransferase (AST) levels higher than the normal range, respectively. Thirty-four percent of patients showed albumin (ALB) levels lower than the normal range, and 6% of patients showed abnormal total bilirubin (TBil) levels. The levels of creatinine (CRE) were increased in 8% of patients. The creatine kinase (CK) level of 13% of patients exceeded the normal range, and 52% of patients had elevated lactate dehydrogenase (LDH) levels. In addition, six studies met the inclusion criteria for the systemic review evaluating the relevance between LDH levels and the severity of COVID-19, and all six studies showed a positive association between these two factors. Conclusions Some patients with COVID-19 had different degrees of blood biochemical abnormalities, which might indicate multiple organ dysfunction. Some biochemical indexes, such as abnormal ALB and LDH, could reflect the severity of the disease to a certain extent. These blood biochemical indicators should be considered in the clinical management of the disease.

Ascitic fluid total protein, a useful marker in non-portal hypertensive ascites.

BACKGROUND AND AIMS: Diagnostic performance of ascitic fluid total protein (AFTP) concentration remained unsettled. Our aim was to determine diagnostic value of AFTP in differential diagnosis of causes of ascites. METHODS: Seven hundred four consecutive patients with new-onset ascites were prospectively enrolled in this study. RESULTS: In the training cohort, diagnostic performance of quantitative AFTP assay was superior to that of Rivalta test in differential diagnosis of ascites. At the predetermined cut-off value of 25 g/L, quantitative AFTP assay was more useful in the differentiation of non-portal hypertensive ascites from portal hypertensive ascites compared with the exudate-transudate classification, area under curve of receiver operating characteristic curve was 0.958. Quantitative AFTP assay was superior to serum-ascites albumin gradient in the detection of non-portal hypertensive ascites, especially malignant ascites and tuberculous peritonitis. In mixed ascites, AFTP was useful in identifying peritoneal lesions. CONCLUSIONS: Ascitic fluid total protein is a useful marker in non-portal hypertensive ascites; thus, it should be determined in diagnostic work-up of the patients with ascites.

Cell-specific elevation of Runx2 promotes hepatic infiltration of macrophages by upregulating MCP-1 in high-fat diet-induced mice NAFLD.

OBJECTIVE: We have demonstrated runt-related transcription factor 2 (Runx2) plays important role in atherosclerosis. It has been indicated that atherosclerosis shares the similar histopathology with nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), on macrophages infiltration. However, the function of Runx2 in NAFLD is completely unknown. Here, we investigated the underlying mechanism of Runx2 triggering macrophages infiltration in the development of NAFLD. METHODS: Mice were fed with high-fat diet (HFD) for a long time. Histopathologic features, macrophages infiltration, expression of monocyte chemotactic protein 1 (MCP-1), and Runx2 were, respectively, analyzed in vivo. Lentivirus or short interfering RNA were transfected in murine hepatic stellate cells (HSCs) and the transwell assay was performed to verify the contribution of Runx2 for macrophages migration in vitro. RESULTS: Long-term treatment with HFD induced the progression of NAFLD, and NASH was initiated from 8 months on diet. HFD increased the expression of F4/80 upon HFD feeding, indicated HFD promotes hepatic infiltration of macrophages in NAFLD. In addition, HFD upregulated the expression of MCP-1 and Runx2 during NAFLD development. Unexpectedly, Runx2 upregulation is cell-type depended in NAFLD, and only abundantly elevated in activated HSCs. Furthermore, we found that Runx2 could increase or decrease the expression of MCP-1 in HSCs, and regulate macrophages migration by influencing MCP-1 production in vitro. CONCLUSIONS: Our results give evidence that the upregulation of Runx2 specific in activated HSCs promotes hepatic infiltration of macrophages by increasing MCP-1 expression in NAFLD, which reveals a novel mechanism and provides a cell-specific therapeutic target for NAFLD.

Regulation of SREBP-2 intracellular trafficking improves impaired autophagic flux and alleviates endoplasmic reticulum stress in NAFLD.

Sterol regulatory element-binding protein 2 (SREBP-2), is a well-known transcriptional regulator of cholesterol metabolism. SREBP-2 is activated unconventionally to promote excessive cholesterol accumulation in non-alcoholic fatty liver disease (NAFLD). In addition, recent studies suggested that excessive lipid and cholesterol accumulation can weaken cellular autophagy function and promote endoplasmic reticulum stress (ERS). However, it remains unknown whether regulation of SREBP-2 processing modulates autophagy and ERS. In this study, we demonstrated that inhibition of SREBP-2 intracellular trafficking by site-1 protease (S1P) and site-2 protease (S2P) specific inhibitors, or shRNAs targeting S1P and S2P, upregulated gene and protein expression of autophagy markers, and improved the impaired autophagic flux induced in both cell and mouse models of NAFLD. Furthermore, increased lipid degradation by autophagy could repress PERK-P-EIF2α signaling. Taken together, these findings suggest that regulating the nuclear transport of SREBP-2 reduces lipid deposition and ERS via an autophagy-dependent pathway.

Evaluation of tumor markers for the differential diagnosis of benign and malignant ascites.

INTRODUCTION: The diagnosis of malignant ascites is a challenging problem in clinical practice, non-invasive techniques should be developed to improve diagnostic accuracy. The diagnostic performances of tumor markers in malignant ascites remained unsettled. Our aim was to evaluate diagnostic performance of tumor markers in differential diagnosis of benign and malignant ascites. MATERIAL AND METHODS: A total of 437 patients were enrolled, and the relevant parameters of the patients were analyzed for the differentiation of benign ascites from malignant ascites. RESULTS: At the predetermined cutoff values of tumor makers, tumor markers in ascitic fluid showed better diagnostic performance than those in serum. Combined use of tumor markers and the cytology increased the diagnostic yield of the latter by 37%. In cytologically negative malignant ascites, tumor markers provided assistance in differentiating malignant ascites from benign ascites, and the combination of ascitic tumor markers yielded 86% sensitivity, 97% specificity. CONCLUSION: Use of a panel of tumor markers exhibited excellent diagnostic performance in diagnosing malignant ascites, which indicated the detection of tumor markers may represent a beneficial adjunct to cytology, thus guiding the selection of patients who might benefit from further invasive procedures.

Efficacy and safety of tauroursodeoxycholic acid in the treatment of liver cirrhosis: a double-blind randomized controlled trial.

No direct comparison of tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) has yet been carried out in the treatment of liver cirrhosis in China. We designed a double-blind randomized trial to evaluate the potential therapeutic efficacy of TUDCA in liver cirrhosis, using UDCA as parallel control. The enrolled 23 patients with liver cirrhosis were randomly divided into TUDCA group (n=12) and UDCA group (n=11), and given TUDCA and UDCA respectively at the daily dose of 750 mg, in a randomly assigned sequence for a 6-month period. Clinical, biochemical and histological features, and liver ultrasonographic findings were evaluated before and after the study. According to the inclusion criteria, 18 patients were included in the final analysis, including 9 cases in both two groups. Serum ALT, AST and ALP levels in TUDCA group and AST levels in UDCA group were significantly reduced as compared with baseline (P<0.05). Serum albumin levels were significantly increased in both TUDCA and UDCA groups (P<0.05). Serum markers for liver fibrosis were slightly decreased with the difference being not significant in either group. Only one patient in TUDCA group had significantly histological relief. Both treatments were well tolerated and no patient complained of side effects. It is suggested that TUDCA therapy is safe and appears to be more effective than UDCA in the treatment of liver cirrhosis, particularly in the improvement of the biochemical expression. However, both drugs exert no effect on the serum markers for liver fibrosis during 6-month treatment.

Exploration of Perturbed Liver Fibrosis-Related Factors and Collagen Type I in Animal Model of Non-Alcoholic Fatty Liver Disease.

To determine their involvement in the onset of the disease, we investigated the changing levels of liver fibrosis-related proteins, namely, type-I collagen, α-smooth muscle actin (α-SMA), and transforming growth factor ß1 and ß3 (TGF-ß1, ß3). The four groups of Sprague-Dawley (SD) rats were involved in the study, namely, (i) normal control group, (ii) high-fat diet group (HFD), (iii) carbon tetrachloride (CCl4) group, and (iv) NAFLD group (animal model) which were chosen at random. The NAFLD model received HFD combined with subcutaneous injection of small doses of CCl4. Histopathological examination confirmed extent of liver fibrosis, while other immunological and molecular methods were used to evaluate expression and distribution of α-SMA, type I collagen TGF-ß1 and TGF-ß3, at both m-RNA and protein levels. In contrast to the normal control group, the NAFLD group showed moderately elevated expressions of TGF-ß1, α-SMA, and type I collagen, which was proportional on temporal scale of NAFLD persistence in the model (P < 0.05). In the early phage of NAFLD, enhancement in the mRNA transcripts and, henceforth, protein expression of TGF-ß3 was observed. However, these were found to be downregulated in case of liver fibrosis (P < 0.05). This NAFLD rat model shows the histopathologic changes of human NAFLD and is suitable for the study of NAFLD pathogenesis. These findings suggest that type I collagen and the liver fibrosis-related factors TGF- ß1, TGF- ß3, and α-SMA may be significant contributors to NAFLD. Although NAFLD model is previously demonstrated by other researchers, our study is novel in terms of exploration of involvement of fibrosis-related factors and in particular aforementioned proteins at the early stage of NAFLD vis-à-vis dynamics of type-I collagen distribution.

Intestinal BMP-9 locally upregulates FGF19 and is down-regulated in obese patients with diabetes.

Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.

[Effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGFb3) mRNA expression and promoter activity in hepatic stellate cells].

OBJECTIVE: To investigate the effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGF b3) mRNA expression and promoter activity in hepatic stellate cells (HSCs). METHODS: Freshly isolated HSCs from rats were divided into six groups: CREB-1 expression plasmid transfected group (C), siRNA-CREB-1 plasmid transfected group (S), negative control group (N), forskolin treated group (F), H-89 treated group (H), and blank group (B). Rats in each group were further sub-divided according to whether (+) or not (-) they were exposed to exogenous TGF b3. TGF b3 mRNA expression was measured by real time quantitative PCR. HSCs of the C, S, N, F, H and B groups were transfected with the TGF b3 promoter luciferase reporter plasmid (PGL3-TGF b3-P; W group), the TGF b3 promoter luciferase reporter plasmid with CRE mutation (PGL3-basic-TGF b3P-mCRE; M group) and the renilla luciferase control plasmid (pRL-SV40; control group). TGF b3 promoter activity was assessed by luciferase reporter assays. RESULTS: Compared to N(-), the TGF b3 mRNA expression was reduced to 0.69+/-0.15 in S(-) (P less than 0.05) and increased to 4.68+/-2.76 in C(-) (P more than 0.05). Compared to B(-), the TGF b3 mRNA expression was reduced to 0.57+/-0.08 in H(-) (P less than 0.05). The differences between N(+) and N(-), S(+) and S(-), B(+) and B(-), and H(+) and H(-) were all significant (P less than 0.05). The values of TGF b3 promoter activity in S(W), N(W), and C(W) were 0.062+/-0.013, 0.122+/-0.011, and 0.165+/-0.016 (P less than 0.05), but the changes of TGF b3 promoter activity in S(M), N(M), and C(M) were not significant (P more than 0.05). The values of TGF b3 promoter activity in H(W), B(W), and F(W) were 0.154+/-0.010, 0.188+/-0.016, and 0.276+/-0.031 (P less than 0.05), but the changes of TGF b3 promoter activity in H(M), B(M), and F(M) were not significant (P more than 0.05). CONCLUSION: Increased levels of CREB-1 mRNA or p-CREB-1 up-regulate the TGF b3 mRNA expression and promoter activity in rat HSCs. The CRE site in the TGF b3 promoter is critical for this effect, and the gene's activity becomes significantly decreased when the site is missing. Exogenous TGF b3 enhances expression of endogenous TGF b3 in rat HSCs.

CREBH alleviates mitochondrial oxidative stress through SIRT3 mediating deacetylation of MnSOD and suppression of Nlrp3 inflammasome in NASH.

Lipotoxicity and unresolved oxidative stress are key drivers of metabolic inflammation in nonalcoholic steatohepatitis (NASH). cAMP-response element binding protein H(CREBH) is a liver-specific transcription factor and regulates the glucose and lipid metabolism of NASH. However, its role in mitochondrial oxidative stress and its association with sirtuin 3 (SIRT3), a master regulator of deacetylation for mitochondrial proteins, remains elusive. In this study, AML-12 cells were treated with palmitic acid to imitate the pathological changes of NASH in vitro and 8-week-old male C57BL/6J mice were fed with a high-fat (HF) diet or a methionine-choline-deficient (MCD) diet to build the widely accepted in vivo model of NASH. We found that lipid overload induced mitochondrial oxidative stress and stimulated the expression of CREBH and SIRT3. CREBH overexpression alleviated the mitochondrial oxidative stress. Moreover, CREBH promoted SIRT3 expression, which regulated the deacetylation of manganese superoxide dismutase (MnSOD) and inhibited NOD-Like Receptor Pyrin Domain Containing 3 (Nlrp3) inflammasome activation whereas suppression of SIRT3 damaged the protecting ability of CREBH in mitochondrial oxidative stress. CREBH knockout mice were highly susceptible to HF and MCD diet-induced NASH with more severe oxidative stress. Collectively, our results firstly provided the support that CREBH could serve as a protective factor in the progression of NASH by regulating the acetylation of MnSOD and the activation of Nlrp3 inflammasome through SIRT3. These results suggest that CREBH might be a valuable therapeutic candidate for NASH.

Difference between type 2 gastroesophageal varices and isolated fundic varices in clinical profiles and portosystemic collaterals.

BACKGROUND: There is significant heterogeneity between gastroesophageal varices (GOV2) and isolated gastric varices (IGV1). The data on the difference between GOV2 and IGV1 are limited. AIM: To determine the etiology, clinical profiles, endoscopic findings, imaging signs, portosystemic collaterals in patients with GOV2 and IGV1. METHODS: Medical records of 252 patients with gastric fundal varices were retrospectively collected, and computed tomography images were analyzed. RESULTS: Significant differences in routine blood examination, Child-Pugh classification and MELD scores were found between GOV2 and IGV1. The incidence of peptic ulcers in patients with IGV1 (26.55%) was higher than that of GOV2 (11.01%), while portal hypertensive gastropathy was more commonly found in patients with GOV2 (22.02%) than in those with IGV1 (3.54%). Typical radiological signs of cirrhotic liver were more commonly observed in patients with GOV2 than in those with IGV1. In patients with GOV2, the main afferent vessels were via the left gastric vein (LGV) (97.94%) and short gastric vein (SGV) (39.18%). In patients with IGV1, the main afferent vessels were via the LGV (75.61%), SGV (63.41%) and posterior gastric vein (PGV) (43.90%). In IGV1 patients with pancreatic diseases, spleno-gastromental-superior mesenteric shunt (48.15%) was a major collateral vessel. In patients with fundic varices, the sizes of gastric/esophageal varices were positively correlated with afferent vessels (LGVs and PGVs) and efferent vessels (gastrorenal shunts). The size of the esophageal varices was negatively correlated with gastrorenal shunts in GOV2 patients. CONCLUSION: Significant heterogeneity in the etiology and vascular changes between GOV2 and IGV1 is useful in making therapeutic decisions.

[Effects of exogenous transforming growth factor-ß3 on the activities of its promoter and cAMP-responsive element binding protein-1 in rat hepatic stellate cell].

OBJECTIVE: To explore the effects of exogenous transforming growth factor-ß3 (TGF-ß3) on the activities of its promoter and cAMP-responsive element binding protein-1 (CREB-1) in rat hepatic stellate cell (HSC-T6). METHODS: HSC-T6 was cultured and treated with or without exogenous TGF-ß3 (10 µg/L). Then cell extracts, total RNA and nuclear proteins were collected at different time points. The specimens were detected by luciferase reporter assay, Western blotting and real-time RT-PCR (reverse transcription-polymerase chain reaction) respectively. RESULTS: After treatment, the activity of TGF-ß3 promoter peaked at 24 h (10.68 ± 0.57 vs 4.83 ± 0.56, 2.2 folds vs control). And the mutational CRE site completely blocked the activity of TGF-ß3 promoter (0.73 ± 0.03, P < 0.05). In addition, exogenous TGF-ß3 increased the expression of phospho-CREB-1 in a time-dependent manner. It peaked at 1 h (2.0 folds vs control) and declined slowly. And exogenous TGF-ß3 had no effect on the mRNA and protein expressions of CREB-1 (P > 0.05). CONCLUSION: The activity of TGF-ß3 promoter is up-regulated by exogenous TGF-ß3. And CRE site in TGF-ß3 promoter region is important for the transcription of TGF-ß3 gene in HSC-T6. While activating CREB-1, exogenous TGF-ß3 has no effect on the expressions of CREB-1 protein and mRNA.

[Effects of exogenous TGF-ß3 on the expression of endogenous TGF-ß3 in hepatic stellate cell-T6 (HSC-T6)].

OBJECTIVE: To investigate the effects of exogenous TGF-ß3 on the expression of endogenous TGF-b3 in hepatic stellate cell (HSC). METHODS: HSCs were cultured and divided into two groups: TGF-ß3 group and blank control group, the cells of TGF-ß3 group were exposed to TGF-b3 (10 ng/ml), whereas the blank control group was not treated. The cells were incubated in the presence of exogenous TGF-ß3 and then (1) were harvested at 0h, 1h, 2h, 4h, 12h, 24h, and real time PCR was performed to detect the mRNA expression of endogenous TGF-ß3. (2) The cells were collected at 0h, 1h, 6h, 12h, and western-blot was used to detect the protein synthesis of endogenous TGF-ß3 in HSC; (3) The cell culture supernatant was harvested at 0h, 1h, 2h, 4h, 8h, 14h, 24h, and ELISA was performed to measure the total protein of extracellular TGF-ß3; HSCs were treated with TGF-ß3 (10 ng/ml) for 2h. The cells were then incubated in serum-free medium and the cell culture supernatant was harvested at 2.25h, 2.5h, 3h, 4h, 6h, 10h and 14h. ELISA was used to detect the extracellular secret ion of endogenous TGF-ß3 by HSCs. RESULTS: (1) Exogenous TGF-ß3 treatment induced a marked increase in TGF-ß3 mRNA expression. By 2h of exogenous TGF-ß3 treatment, maximal TGF-ß3 mRNA expression levels (2.796 ± 0.518) of 2.74 fold above control values (1.022 ± 0.038) was reached (P < 0.05). Thereafter, TGF-ß3 mRNA expression level declined, and the expression level was maintained at level of 1.45-fold for at least 10h and was 1.18-fold above control values by 24h TGF-ß3 treatment (P < 0.05); (2) No significant difference about the intracellular protein expression level of endogenous TGF-ß3 was found between two groups. (P > 0.05); (3) The total expression level of TGF-ß3 reached a peak [(18.931 ± 2.904) ng/ml] at 4h after TGF-ß3 treatment (1.89-fold higher than basic TGF-ß3 (10 ng/ml). After that, it slowly declined. The expression peak [(0.835 ± 0.027) ng/ml] induction of extracellular secreted TGF-ß3 was at 3h (32.12-fold higher than control [(0.026 ± 0.022) ng/ml], (P < 0.05). Thereafter, TGF-ß3 slowly decreased after the peak time, and their expressions were still statistically significant as compared to the control (P < 0.05). CONCLUSION: Exogenous TGF-ß3 could increase the expression of endogenous TGF-ß3 mRNA and extracellular secreted TGF-ß3 protein obviously.

Zhikang Capsule Ameliorates Inflammation, Drives Polarization to M2 Macrophages, and Inhibits Apoptosis in Lipopolysaccharide-induced RAW264.7 Cells.

OBJECTIVE: To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule (ZKC) on lipopolysaccharide (LPS)-induced RAW264.7 cells. METHODS: Safe concentrations of ZKC (0.175, 0.35, and 0.7 mg/mL) were used after the half-maximal inhibitory concentration (IC50) of RAW264.7 cells was calculated through the CCK-8 assay. In addition, the optimal intervention duration of ZKC (0.7 mg/mL) on RAW264.7 cells was determined to be 6 h, since all proinflammatory mediators [tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), inteleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and monocyte chemotactic protein-1 (MCP-1)] had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations (4, 8, and 12 h). RAW264.7 cells were pretreated with ZKC at various concentrations (0.175, 0.35 and 0.7 mg/mL) for 6 h and then stimulated with LPS (1 µg/mL) for an additional 12 h. RESULTS: In terms of inflammation, ZKC could reverse LPS-induced upregulation of TNF-α, IL-1ß, IL-6, COX-2, iNOS, and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner. In terms of the NF-κB signaling pathway, ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner. Moreover, ZKC exhibited a protective effect on macrophages from apoptosis. CONCLUSION: ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level, and a weakened NF-κB signaling pathway may be a potential significant target.

Resolvin D1 attenuates CCl4 Induced Liver Fibrosis by Inhibiting Autophagy-Mediated HSC activation via AKT/mTOR Pathway.

Resolvin D1 (RvD1) was previously reported to relieve inflammation and liver damage in several liver diseases, but its potential role in liver fibrosis remains elusive. The aim of our study was to investigate the effects and underlying mechanisms of RvD1 in hepatic autophagy in liver fibrosis. In vivo, male C57BL/6 mice were intraperitoneally injected with 20% carbon tetrachloride (CCl4, 5 ml/kg) twice weekly for 6 weeks to establish liver fibrosis model. RvD1 (100 ng or 300 ng/mouse) was added daily in the last 2 weeks of the modeling period. In vitro, lipopolysaccharide (LPS)-activated LX-2 cells were co-treated with increasing concentrations (2.5-10 nM) of RvD1. The degree of liver injury was measured by detecting serum AST and ALT contents and H&E staining. Hepatic fibrosis was assessed by masson's trichrome staining and metavir scoring. The qRT-PCR, western blot, immunohistochemistry, and immunofluorescence were applied to liver tissues or LPS-activated LX-2 cells to explore the protective effects of RvD1 in liver fibrosis. Our findings reported that RvD1 significantly attenuated CCl4 induced liver injury and fibrosis by decreasing plasma AST and ALT levels, reducing collagen I and α-SMA accumulation and other pro-fibrotic genes (CTGF, TIMP-1 and Vimentin) expressions in mouse liver, restoring damaged histological architecture and improving hepatic fibrosis scores. In vitro, RvD1 also repressed the LPS induced LX-2 cells activation and proliferation. These significant improvements mainly attributed to the inhibiting effect of RvD1 on autophagy in the process of hepatic stellate cell (HSC) activation, as demonstrated by decreased ratio of LC3-II/I and elevated p62 after RvD1 treatment. In addition, using AZD5363 (an AKT inhibitor that activates autophagy) and AZD8055 (an mTOR inhibitor, another autophagy activator), we further verified that RvD1 suppressed autophagy-mediated HSC activation and alleviated CCl4 induced liver fibrosis partly through AKT/mTOR pathway. Overall, these results demonstrate that RvD1 treatment is expected to become a novel therapeutic strategy against liver fibrosis.