Yuan-Wen Chen

Cardiac Kallikrein-Kinin System Is Upregulated in Chronic Volume Overload and Mediates an Inflammatory Induced Collagen Loss

Background The clinical problem of a “pure volume overload” as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat. Methodology/Principal Findings LV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK2R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK2R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK2R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin. Conclusions/Significance A pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction–all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

Total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota

Non-alcoholic steatohepatitis (NASH) is an epidemic metabolic disease with limited therapeutic strategies. Cumulative data support the pivotal role of gut microbiota in NASH. Here, we investigated the hypothesis regarding whether fecal microbiota transplantation (FMT) is effective in attenuating high-fat diet (HFD)-induced steatohepatitis in mice. Mice were randomized into control, HFD and HFD + FMT groups. After an 8-week HFD, FMT treatment was initiated and carried out for 8 weeks. The gut microbiota structure, butyrate concentrations of the cecal content, liver pathology and intrahepatic lipid and cytokines were examined. Our results showed that after FMT, the gut microbiota disturbance was corrected in HFD-fed mice with elevated abundances of the beneficial bacteria Christensenellaceae and Lactobacillus. FMT also increased butyrate concentrations of the cecal content and the intestinal tight junction protein ZO-1, resulting in relief of endotoxima in HFD-fed mice. Steatohepatitis was alleviated after FMT, as indicated by a significant decrease in intrahepatic lipid accumulation (reduced Oli-red staining, decreased intrahepatic triglyceride and cholesterol), intrahepatic pro-inflammatory cytokines, and the NAS score. Accordingly, intrahepatic IFN-γ and IL-17 were decreased, but Foxp3, IL-4 and IL-22 were increased after FMT intervention. These data indicate that FMT attenuated HFD-induced steatohepatitis in mice via a beneficial effect on the gut microbiota.

Disease-specific miR-34a as diagnostic marker of non-alcoholic steatohepatitis in a Chinese population

AIM To assess disease-specific circulating microRNAs (miRNAs) in non-alcoholic steatohepatitis (NASH) patients. METHODS A total of 111 biopsy-proven non-alcoholic fatty liver disease (NAFLD) or chronic hepatitis B (CHB) patients and healthy controls from mainland China were enrolled to measure their serum levels of miR-122, -125b, -146b, -16, -21, -192, -27b and -34a. The correlations between serum miRNAs and histological features of NAFLD were determined. The diagnostic value of miRNA in NASH and significant fibrosis was analyzed and compared with that of cytokeratin-18 (CK-18), fibrosis-4 (FIB-4), and aspartate aminotransferase to platelet ratio index (APRI), respectively. RESULTS Circulating miR-122, -16, -192 and -34a showed differential expression levels between NAFLD and CHB patients, and miR-34a had an approximately 2-fold increase in NAFLD samples compared with that of CHB samples (P < 0.01). Serum miR-122, -192 and -34a levels were correlated with steatosis (R = 0.302, 0.323 and 0.470, respectively, P < 0.05) and inflammatory activity (R = 0.445, 0.447 and 0.517, respectively, P < 0.01); only serum miR-16 levels were associated with fibrosis (R = 0.350, P < 0.05) in patients with NAFLD. The diagnostic value of miR-34a for NASH (area under the receiver operating characteristic, 0.811, 95%CI: 0.670-0.953) was superior to that of alanine aminotransferase, CK-18, FIB-4 and APRI in NAFLD, but miR-16 showed a limited performance in the diagnosis of significant fibrosis in NASH. CONCLUSION Circulating miR-34a may serve as a disease-specific noninvasive biomarker for the diagnosis of NASH.

Sodium butyrate attenuates high-fat diet-induced steatohepatitis in mice by improving gut microbiota and gastrointestinal barrier.

AIM To investigate whether gut microbiota metabolite sodium butyrate (NaB) is an effective substance for attenuating non-alcoholic fatty liver disease (NAFLD) and the internal mechanisms. METHODS Male C57BL/6J mice were divided into three groups, normal control were fed standard chow and model group were fed a high-fat diet (HFD) for 16 wk, the intervention group were fed HFD for 16 wk and treated with NaB for 8 wk. Gut microbiota from each group were detected at baseline and at 16 wk, liver histology were evaluated and gastrointestinal barrier indicator such as zonula occluden-1 (ZO-1) were detected by immunohistochemistry and realtime-PCR, further serum or liver endotoxin were determined by ELISA and inflammation- or metabolism-associated genes were quantified by real-time PCR. RESULTS NaB corrected the HFD-induced gut microbiota imbalance in mice, while it considerably elevated the abundances of the beneficial bacteria Christensenellaceae, Blautia and Lactobacillus. These bacteria can produce butyric acid in what seems like a virtuous circle. And butyrate restored HFD induced intestinal mucosa damage, increased the expression of ZO-1 in small intestine, further decreased the levels of gut endotoxin in serum and liver compared with HF group. Endotoxin-associated genes such as TLR4 and Myd88, pro-inflammation genes such as MCP-1, TNF-α, IL-1, IL-2, IL-6 and IFN-γ in liver or epididymal fat were obviously downregulated after NaB intervention. Liver inflammation and fat accumulation were ameliorated, the levels of TG and cholesterol in liver were decreased after NaB intervention, NAS score was significantly decreased, metabolic indices such as FBG and HOMA-IR and liver function indicators ALT and AST were improved compared with HF group. CONCLUSION NaB may restore the dysbiosis of gut microbiota to attenuate steatohepatitis, which is suggested to be a potential gut microbiota modulator and therapeutic substance for NAFLD.

Matrix metalloproteinase 2 contributes to pancreatic Beta cell injury induced by oxidative stress.

Objective To investigate the role of matrix metalloproteinase 2 (MMP2) in pancreatic beta cell injury induced by oxidative stress. Methods Rat pancreatic beta cell line INS-1 cells were treated with advanced glycation end-products (AGE) to induce intracellular oxidative stress. Intracellular MMP2 expression and activity were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and zymography, respectively. MMP2 expression and activity were manipulated by over-expression with recombinant MMP2 plasmids or knockdown with either MMP2 specific siRNA or inhibitors, and effects on apoptosis and insulin-secretion were measured by flow cytometry and ELISA. Results AGE treatment induced intracellular oxidative stress in INS-1 cells, as indicated by elevated ROS levels, apoptotic cell death, and suppressed insulin secretion. This was accompanied by increased MMP2 expression and activity. However, Antioxidant N-acetylcysteine (NAC) treatment inhibited MMP2 expression and activity, and partially reversed cell apoptosis and insulin secretion dysfunction induced by AGE. Forced expression of MMP2 mimicked the effects of AGE treatment while inhibition of MMP2 either by a specific MMP2 inhibitor or MMP2 siRNA protected oxidative stress induced by AGE. Conclusion MMP2 expression and intracellular activity are increased by oxidative stress, contributing to cellular dysfunction and apoptosis in INS-1 cells after AGE challenge.

Steatosis induced CCL5 contributes to early-stage liver fibrosis in nonalcoholic fatty liver disease progress

&NA; The rapidly increasing prevalence of nonalcoholic fatty liver disease (NAFLD) has become one of the major public health threats in China and worldwide. However, during the development of NAFLD, the key mechanism underlying the progression of related fibrosis remains unclear, which greatly impedes the development of optimal NAFLD therapy. In the current study, we were endeavored to characterize a proinflammatory cytokine, CCL5, as a major contributor for fibrosis in NAFLD. The results showed that CCL5 was highly expressed in fatty liver and NASH patients. In NAFLD rats induced by 8‐week‐HFD, CCL5 and its receptor, CCR5, were significantly up‐regulated and liver fibrosis exclusively occurred in this group. In addition, we showed that hepatocytes are the major source contributing to this CCL5 elevation. Interestingly, a CCL5 inhibitor Met‐CCL5, significantly decreased liver fibrosis but not hepatic steatosis. Using a cell model of hepatic steatosis, we found that the conditioned medium of lipid‐overloaded hepatocytes (Fa2N‐4 cells) which produced excessive CCL5 stimulated the profibrotic activities of hepatic stellate cells (LX‐2) as manifested by increased migration rate, proliferation and collagen production of LX‐2 cells. CCL5 knockdown in Fa2N‐4 cells, Met‐CCL5 or CCR5 antibody treatment on LX‐2 cells all significantly inhibited the conditioned medium of FFA‐treated Fa2N‐4 cells to exert stimulatory effects on LX‐2 cells. Consistently, the conditioned medium of Fa2N‐4 cells with CCL5 over‐expression significantly enhanced migration rate, cell proliferation and collagen production of LX‐2 cells. All these results support that CCL5 produced by steatotic hepatocytes plays an essential role in fibrotic signaling machinery of NAFLD. In addition, we were able to identify C/EBP‐&bgr; as the up‐stream regulator of CCL5 gene transcription in hepatocytes treated with free fatty acid (FFA). Our data strongly supported that CCL5 plays a pivotal regulatory role in hepatic fibrosis during NAFLD, which constitutes a novel and exciting observation that may call for potential future development of specific CCL5‐targeted NAFLD therapy.

Comparison of Porcine Small Intestinal Submucosa versus Polypropylene in Open Inguinal Hernia Repair: A Systematic Review and Meta-Analysis

Background A systematic review and meta-analysis was performed in randomized controlled trials (RCTs) to compare porcine small intestinal submucosa (SIS) with polypropylene in open inguinal hernia repair. Method Electronic databases MEDLINE, Embase, and the Cochrane Library were used to compare patient outcomes for the two groups via meta-analysis. Result A total of 3 randomized controlled trials encompassing 200 patients were included in the meta-analysis. There was no significant difference in recurrence (P = 0.16), hematomas (P = 0.06), postoperative pain within 30 days (P = 0.45), or postoperative pain after 1 year (P = 0.12) between the 2 groups. The incidence of discomfort was significantly lower (P = 0.0006) in the SIS group. However, the SIS group experienced a significantly higher incidence of seroma (P = 0.03). Conclusions Compared to polypropylene, using SIS in open inguinal hernia repair is associated with a lower incidence of discomfort and a higher incidence of seroma. However, well-designed larger RCT studies with a longer follow-up period are needed to confirm these findings.

Clostridium butyricum B1 alleviates high-fat diet-induced steatohepatitis in mice via enterohepatic immunoregulation.

Enterohepatic immunologic derangement is associated with non‐alcoholic steatohepatitis. Here, we investigated whether Clostridium butyricum B1 (CB) would be an effective immune‐targeted substance to attenuate steatohepatitis in mice.

Maternal high-fat diet during pregnancy and lactation affects hepatic lipid metabolism in early life of offspring rat

We investigated whether maternal over-nutrition during pregnancy and lactation affects the offspring’s lipid metabolism at weaning by assessing liver lipid metabolic gene expressions and analysing its mechanisms on the development of metabolic abnormalities. Female Sprague–Dawley rats were fed with standard chow diet (CON) or high-fat diet (HFD) for 8 weeks, and then continued feeding during gestation and lactation. The offspring whose dams were fed with HFD had a lower birth weight but an increased body weight with impaired glucose tolerance, higher serum cholesterol, and hepatic steatosis at weaning. Microarray analyses showed that there were 120 genes differently expressed between the two groups. We further verified the results by qRT-PCR. Significant increase of the lipogenesis (Me1, Scd1) gene expression was found in HFD (P<0.05), and up-regulated expression of genes (PPAR-α, Cpt1α, Ehhadh) involved in β-oxidation was also observed (P<0.05), but the Acsl3 gene was down-regulated (P<0.05). Maternal over-nutrition could not only primarily induce lipogenesis, but also promote lipolysis through an oxidation pathway as compensation, eventually leading to an increased body weight, impaired glucose tolerance, elevated serum cholesterol and hepatic steatosis at weaning. This finding may provide some evidence for a healthy maternal diet in order to reduce the risk of metabolic diseases in the early life of the offspring.

Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line

Background Prolyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis. Methods The human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor) at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot. Results Compared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 μM) incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA)-treated cells at 26–130 μM; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026–65 μM without FFA treatment. S17092 treatment (13 and 26 μM) also elicited no significant effect on apoptosis in normal L02 cells, but FFA treatment increased cell apoptosis, which was attenuated by S17092 incubation. S17092 treatment inhibited intracellular POP activity and decreased the AcSDKP level at the concentration of 0.026–26 μM. After treatment with FFA for 24 h, oil red O staining revealed significant lipid accumulation in the cells in the model group compared with the controls; however, lipid accumulation was suppressed after the administration of S17092 (13 and 26 μM). Accordingly, the triglyceride levels in the FFA-treated cells were approximately 5-fold greater than those of the controls and were decreased by approximately 25% and 45% after the administration of S17092 at 13 and 26 μM, respectively. The mRNA levels of FASN, PPAR-γ, and SREBP-1c were higher in the FFA-treated cells than in the normal controls, and all of these levels were significantly inhibited in the presence of S17092 at both 13 and 26 μM. S17092 treatment did not affect LC3B II in the FFA-treated cells compared with FFA treatment alone. Conclusion The expression of POP increases with hepatocyte steatosis, and POP inhibitors can significantly reduce intracellular lipid accumulation, which might be related to the inhibition of genes involved in lipid synthesis.