Involvement of YTHDF1 in renal fibrosis progression via up-regulating YAP.

Renal fibrosis is a progressive, fatal renal disease characterized by the aberrant accumulation of myofibroblasts that produce excess extracellular matrix (ECM) in the renal interstitium and glomeruli. Yes-associated protein (YAP) has been regarded as a crucial modulator in myofibroblast transformation, but its upstream regulator remains a mystery. In the present study investigating the participation of m6A methylation during renal fibrosis through bioinformatics analysis, we identified YTHDF1, a modulator of m6A methylation, as a key contributor for renal fibrosis because it was highly expressed in human fibrotic kidneys and had a significant correction with YAP. Their co-localization in human fibrotic kidneys was additionally shown by immunofluorescence. We then found that YTHDF1 was also up-regulated in fibrotic mouse kidneys induced by unilateral ureteral obstruction (UUO), high-dose folic acid administration, or the unilateral ischemia-reperfusion injury, further supporting a causal role of YTHDF1 during renal fibrosis. Consistent with this notion, YTHDF1 knockdown alleviated the progression of renal fibrosis both in cultured cells induced by transforming growth factor-beta administration and in the UUO mouse model. Meanwhile, YAP was accordingly down-regulated when YTHDF1 was inhibited. Furthermore, the specific binding of YTHDF1 to YAP mRNA was detected using RNA Binding Protein Immunoprecipitation, and the up-regulation of fibrotic related molecules in cultured cells induced by YTHDF1 over-expression plasmid was attenuated by YAP siRNA. Taken together, our data highlight the potential utility of YTHDF1 as an indicator for renal fibrosis and suggest that YTHDF1 inhibition might be a promising therapeutic strategy to alleviate renal fibrosis via downregulating YAP.

ß-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of ß-catenin, one with ß-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived ß-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of ß-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of ß-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or ß-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel ß-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

The liver has an incredible capacity to repair itself or 'regenerate' ­ that is, it has the ability to replace damaged tissue with new tissue. In order to do this, the organ relies on hepatocytes (the cells that form the liver) and bile duct cells (the cells that form the biliary ducts) dividing and transforming into each other to repair and replace damaged tissue, in case the insult is dire. During long-lasting or chronic liver injury, bile duct cells undergo a process called 'ductular reaction', which causes the cells to multiply and produce proteins that stimulate inflammation, and can lead to liver scarring (fibrosis). Ductular reaction is a hallmark of severe liver disease, and different diseases exhibit ductular reactions with distinct features. For example, in cystic fibrosis, a unique type of ductular reaction occurs at late stages, accompanied by both inflammation and fibrosis. Despite the role that ductular reaction plays in liver disease, it is not well understood how it works at the molecular level. Hu et al. set out to investigate how a protein called ß-catenin ­ which can cause many types of cells to proliferate ­ is involved in ductular reaction. They used three types of mice for their experiments: wild-type mice, which were not genetically modified; and two strains of genetically modified mice. One of these mutant mice did not produce ß-catenin in biliary duct cells, while the other lacked ß-catenin both in biliary duct cells and in hepatocytes. After a short liver injury ­ which Hu et al. caused by feeding the mice a specific diet ­ the wild-type mice were able to regenerate and repair the liver without exhibiting any ductular reaction. The mutant mice that lacked ß-catenin in hepatocytes showed a temporary ductular reaction, and ultimately repaired their livers by turning bile duct cells into hepatocytes. On the other hand, the mutant mice lacking ß-catenin in both hepatocytes and bile duct cells displayed sustained ductular reactions, inflammation and fibrosis, which looked like that seen in patients with liver disease associated to cystic fibrosis. Further probing showed that ß-catenin interacts with a protein called CTFR, which is involved in cystic fibrosis. When bile duct cells lack either of these proteins, another protein called NF-B gets activated, which causes the ductular reaction, leading to inflammation and fibrosis. The findings of Hu et al. shed light on the role of ß-catenin in ductular reaction. Further, the results show a previously unknown interaction between ß-catenin, CTFR and NF-B, which could lead to better treatments for cystic fibrosis in the future.

Hirsutella sinensis fungus improves cardiac function in mouse model of heart failure.

Cordyceps sinensis, including Hirsutella sinensis, is a highly valuable traditional Chinese medicine and is used to treat patients with pulmonary heart disease in clinical practice. However, the underlying mechanisms of its effects remain unclear. In this study, a mouse model of heart failure established by non-thoracic, transverse aortic constriction (TAC) was developed to determine the underlying mechanisms of therapeutic effects of Hirsutella sinensis fungus (HSF) powder. The results showed that HSF treatment remarkably ameliorated myocardial hypertrophy, collagen fiber hyperplasia, and cardiac function in mice with heart failure. Using transcriptional and epigenetic analyses, we found that the mechanism of HSF mainly involved a variety of signaling pathways related to myocardial fibrosis and determined that HSF could reduce the levels of TGF-ß1 proteins in heart tissue, as well as type I and III collagen levels. These data suggest that HSF alleviates heart failure, inhibits irreversible ventricular remodeling, and improves cardiac function through the regulation of myocardial fibrosis-related signaling pathways, which can provide novel opportunities to improve heart failure therapy.

Parthenolide plays a protective role in the liver of mice with metabolic dysfunction­associated fatty liver disease through the activation of the HIPPO pathway.

Metabolic dysfunction­associated fatty liver disease (MAFLD) is a serious threat to human health. Parthenolide (PAR) displays several important pharmacological activities, including the promotion of liver function recovery during hepatitis. The aim of the present study was to assess the effect of PAR on MAFLD in a mouse model. Body weight, liver to body weight ratios, histological score, alanine transaminase, aspartate transaminase, total cholesterol and triglyceride levels were determined to evaluate liver injury. Liver hydroxyproline concentrations were also assessed. The expression levels of lipid metabolism­related genes (sterol regulatory element binding protein­1c, fatty acid synthase, acetyl CoA carboxylase 1, stearoyl CoA desaturase 1 and carbohydrate response element­binding protein, peroxisome proliferator­activated receptor α, carnitine palmitoyl transferase 1α and acyl­CoA dehydrogenase short chain), liver fibrosis­associated genes (α­smooth muscle actin, tissue inhibitor of metalloproteinase 1 and TGF­ß1), pro­inflammatory cytokines (TNF­α, IL­1ß and IL­6) and oxidative stress­associated enzymes (malondialdehyde, superoxide dismutase and glutathione peroxidase) were measured in mice with MAFLD. The expression levels of genes associated with the HIPPO pathway were also measured. In vivo experiments using a specific inhibitor of HIPPO signalling were performed to verify the role of this pathway in the effects of PAR. PAR exerted beneficial effects on liver injury, lipid metabolism, fibrosis, inflammation and oxidative stress in mice with MAFLD, which was mediated by activation of the HIPPO pathway.

Periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation in mice.

Periplocymarin is an effective component of Periplocae Cortex, which was wildly used as an ingredient in Traditional Chinese Medicine. Our group previously reported that periplocymarin exerted cardiotonic role via promoting calcium influx. However, its exact role in the pathogenesis of myocardial fibrosis has not been elucidated yet. The present study was aimed at determining the potential effect and underlying mechanism of periplocymarin in isoproterenol (ISO)-induced myocardial fibrosis. C57BL/6 mice were subcutaneously injected with ISO (5 mg/kg/day) or saline for 1 week. The early-to-atrial wave ratio (E/A ratio) measured by echocardiography revealed that ISO-induced heart stiffness was remarkably reversed by administration of periplocymarin (5 mg/kg/day). Masson trichrome staining exhibited that treatment of periplocymarin reduced the excessive deposition of extracellular matrix (ECM). Further investigations employing real-time PCR and western blot demonstrated that periplocymarin suppressed the expression of fibrosis related genes (Col1a1, Col3a1, Acta2 and Tgfb1) and proteins (Collagen I, Collagen III, α-SMA and TGF-ß1) induced by ISO. Metabolomics analysis demonstrated that periplocymarin ameliorated the disorders triggered by ISO and many of the differential metabolic substances were involved in amino acid, glucose and lipid metabolism. Further analysis using network pharmacology revealed that three key genes, namely NOS2, NOS3 and Ptgs2, may be the potential targets of periplocymarin and responsible for the disorders. Validation using heart tissues showed that the mRNA expression of NOS3 was decreased while Ptgs2 was increased upon ISO treatment, which were reversed by periplocymarin. Moreover, the expression of COX-2 (Ptgs2 encoded protein) was consistent with the aspect of Ptgs2 mRNA, while eNOS (NOS3 encoded protein) expression was unchanged. In vitro studies exhibited that periplocymarin exerts anti-fibrotic function via regulating at least eNOS and COX-2 in cardiomyocyte. Taken together, periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation, the potential mechanism was that periplocymarin targeted on, at least eNOS and COX-2, to improve the metabolic processes of cardiomyocyte and thus attenuated the myocardial fibrosis. Our study highlighted that periplocymarin is a potential therapeutic agent for the prevention of myocardial fibrosis.

Oligo-Fucoidan Improves Diabetes-Induced Renal Fibrosis via Activation of Sirt-1, GLP-1R, and Nrf2/HO-1: An In Vitro and In Vivo Study.

Fucoidan extracted from brown algae has multiple beneficial functions. In this study, we investigated the effects of low-molecular-weight fucoidan (oligo-FO) on renal fibrosis under in vitro and in vivo diabetic conditions, and its molecular mechanisms. Advanced glycation product (AGE)-stimulated rat renal proximal tubular epithelial cells (NRK-52E) and diabetic mice induced by high-fat diet and intraperitoneal injection of streptozotocin and nicotinamide were used. Oligo-FO treatment significantly inhibited anti-high mobility group box 1 (HMGB1)/RAGE/ anti-nuclear factor-kappa B (NF-κB)/transforming growth factor-ß1 (TGF-ß1)/TGF-ß1R/Smad 2/3/fibronectin signaling pathway and HIF-1α activation in AGE-stimulated NRK-52E cells. Conversely, the expression and activity of Sirt-1; the levels of ubiquitin-specific peptidase 22 (USP22), p-AMPK, glucagon-like peptide-1 receptor (GLP-1R), and heme oxygenase-1 (HO-1); and Nrf2 activation were remarkably increased by oligo-FO in AGE-stimulated cells. However, the above effects of oligo-FO were greatly diminished by inhibiting Sirt-1, HO-1, or GLP-1R activity. Similar changes of these pro-fibrotic genes in the kidney and a marked attenuation of renal injury and dysfunction were observed in oligo-FO-treated diabetic mice. These findings indicated that the inhibitory effects of the oligo-FO on diabetes-evoked renal fibrosis are mediated by suppressing TGF-ß1-activated pro-fibrogenic processes via Sirt-1, HO-1, and GLP-1R dependence. Collectively, fucoidan-containing foods or supplements may be potential agents for ameliorating renal diseases due to excessive fibrosis.

Shenkang VII Recipe Attenuates Unilateral Ureteral Obstruction-induced Renal Fibrosis via TGF-ß/Smad, NF-κB and SHH Signaling Pathway.

This study aimed to explore the protective effects of the traditional Chinese Medicine formula Shenkang VII recipe (SK-7) on renal fibrosis and the mechanisms. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) in rats. The rats were then divided into 5 groups: control group (Sham operation), UUO model group, UUO model plus low to high doses of SK-7 (0.5, 1.0, or 2.0 g/kg/day, for 14 days) groups. The animals were sacrificed on the 7th or 14th day. Kidney tissues were collected for histopathological examinations (hematoxylin and eosin and Masson's trichrome staining). Immunohistochemistry was used to detect the expression of collagen type III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), TIMP metallopeptidase inhibitor 2 (TIMP2), matrix metallopeptidase 2 (MMP2), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and monocyte chemotactic protein-1 (MCP-1). The TGF-ß1/Smad, NF-kB and Sonic hedgehog signaling proteins were detected by Western blotting. Our results showed that SK-7 prevented UUO-induced renal injury and accumulation of collagen fibrils. Renal fibrosis biomarkers Col III, FN, α-SMA and TIMP2 were increased in the rats after UUO and decreased by SK-7, while MMP2 was upregulated after treatment. SK-7 also suppressed the levels of TNF-α, IL-1ß and MCP-1 in UUO rats. In addition, SK-7 inhibited activation of the TGF-ß/Smad, NF-κB and sonic hedgehog signaling (SHH) pathways. Taken together, these findings suggest that SK-7 may regulate the synthesis and degradation of extracellular matrix, reduce inflammation and suppress the proliferation of fibroblasts, by blocking the TGF-ß1/Smad, NF-κB and SHH signaling pathways to exert its anti-renal fibrosis effect in UUO rats.

Vitamin D ameliorates adipose browning in chronic kidney disease cachexia.

Patients with chronic kidney disease (CKD) are often 25(OH)D3 and 1,25(OH)2D3 insufficient. We studied whether vitamin D repletion could correct aberrant adipose tissue and muscle metabolism in a mouse model of CKD-associated cachexia. Intraperitoneal administration of 25(OH)D3 and 1,25(OH)2D3 (75 µg/kg/day and 60 ng/kg/day respectively for 6 weeks) normalized serum concentrations of 25(OH)D3 and 1,25(OH)2D3 in CKD mice. Vitamin D repletion stimulated appetite, normalized weight gain, and improved fat and lean mass content in CKD mice. Vitamin D supplementation attenuated expression of key molecules involved in adipose tissue browning and ameliorated expression of thermogenic genes in adipose tissue and skeletal muscle in CKD mice. Furthermore, repletion of vitamin D improved skeletal muscle fiber size and in vivo muscle function, normalized muscle collagen content and attenuated muscle fat infiltration as well as pathogenetic molecular pathways related to muscle mass regulation in CKD mice. RNAseq analysis was performed on the gastrocnemius muscle. Ingenuity Pathway Analysis revealed that the top 12 differentially expressed genes in CKD were correlated with impaired muscle and neuron regeneration, enhanced muscle thermogenesis and fibrosis. Importantly, vitamin D repletion normalized the expression of those 12 genes in CKD mice. Vitamin D repletion may be an effective therapeutic strategy for adipose tissue browning and muscle wasting in CKD patients.

Bixin Confers Prevention against Ureteral Obstruction-Caused Renal Interstitial Fibrosis through Activation of the Nuclear Factor Erythroid-2-Related Factor2 Pathway in Mice.

Bixin is a natural carotenoid isolated from the seeds of Bixa orellana, with numerous important pharmacological activities, including antioxidant and antifibrotic effects. The nuclear factor erythroid-2-related factor2 (Nrf2) signaling pathway induced by bixin is involved in the process. Excessive reactive oxygen species generation in tubular cells contributes to kidney interstitial fibrosis. The potential therapeutic strategy for bixin in alleviating kidney fibrosis remains largely unclear. In this study, we used unilateral ureteral obstruction (UUO) to establish a renal fibrotic model. Dramatic oxidative DNA damage occurs in kidneys, especially in tubular cells after UUO. In cultured tubular cells, bixin could induce Nrf2 signaling activation by suppressing Nrf2 ubiquitination and increasing its protein stability. Transforming growth factor beta 1-induced epithelial-to-mesenchymal transition (EMT) and extracellular matrix production were suppressed by bixin, and blockade of Nrf2 activation by small interfering RNA could largely reverse the protective effect of bixin. In vivo studies showed that administration of bixin induces Nrf2 signaling activation in tubular cells and markedly attenuates partial EMT of tubular cells and kidney interstitial fibrosis after subjecting to UUO. Together, this study implies that bixin may protect against kidney interstitial fibrosis through stimulating Nrf2 activation in renal tubular cells.

Transcriptomic analysis of the mechanisms of alleviating renal interstitial fibrosis using the traditional Chinese medicine Kangxianling in a rat model.

Renal interstitial fibrosis (RIF) is currently recognized as a crucial mechanism of the pathogenesis of chronic kidney disease (CKD). Kangxianling (KXL, anti-fibrin) is a traditional Chinese medicine that has been proven to significantly reduce the levels of ECM deposition and inhibit renal fibrosis. To characterize the mechanisms and drug targets of KXL, we established a RIF rat model and treated the rats with KXL and losartan. Histological analyses validated the establishment of the RIF model and the treatment effect of KXL. Multiple levels of transcriptomic datasets were generated using lncRNA, mRNA and microRNA sequencing of kidney tissues. Functional annotations and pathway analyses were performed to unravel the therapeutic mechanisms. A multi-level transcriptomic regulatory network was built to illustrate the core factors in fibrosis pathogenesis and therapeutic regulation. KXL and losartan significantly reduced the progression of RIF, and a better therapeutic effect was shown with higher concentrations of KXL. According to the cluster analysis results of the RNA-seq data, the normal control (NC) and high concentration of KXL (HK) treatment groups were the closest in terms of differentially expressed genes. The WNT, TGF-ß and MAPK pathways were enriched and dominated the pathogenesis and therapy of RIF. miR-15b, miR-21, and miR-6216 were upregulated and miR-107 was downregulated in the fibrosis model. These small RNAs were shown to play critical roles in the regulation of the above fibrosis-related genes and could be inhibited by KXL treatment. Finally, based on the lncRNA datasets, we constructed a mRNA-lncRNA-miRNA coexpression ceRNA network, which identified key regulatory factors in the pathogenesis of kidney fibrosis and therapeutic mechanisms of KXL. Our work revealed the potential mechanism of the Chinese medicine Kangxianling in inhibiting renal interstitial fibrosis and supported the clinical use of KXL in the treatment of kidney fibrosis.

Protein hydrolysate from potato confers hepatic-protection in hamsters against high fat diet induced apoptosis and fibrosis by suppressing Caspase-3 and MMP2/9 and by enhancing Akt-survival pathway.

BACKGROUND: A potato protein hydrolysate, APPH is a potential anti-obesity diet ingredient. Since, obesity leads to deterioration of liver function and associated liver diseases, in this study the effect of APPH on high fat diet (HFD) associated liver damages was investigated. METHODS: Six week old male hamsters were randomly separated to six groups (n = 8) as control, HFD (HFD fed obese), L-APPH (HFD + 15 mg/kg/day of APPH), M-APPH (HFD + 30 mg/kg/day), H-APPH (HFD + 75 mg/kg/day of APPH) and PB (HFD + 500 mg/kg/day of probucol). HFD fed hamsters were administered with APPH 50 days through oral gavage. The animals were euthanized and the number of apoptotic nuclei in liver tissue was determined by TUNEL staining and the extent of interstitial fibrosis was determined by Masson's trichrome staining. Modulation in the molecular events associated with apoptosis and fibrosis were elucidated from the western blotting analysis of the total protein extracts. RESULTS: Hamsters fed with high fat diet showed symptoms of liver damage as measured from serum markers like alanine aminotransferase and aspartate aminotransferase levels. However a 50 day long supplementation of APPH effectively ameliorated the effects of HFD. HFD also modulated the expression of survival and apoptosis proteins in the hamster liver. Further the HFD groups showed elevated levels of fibrosis markers in liver. The increase in fibrosis and apoptosis was correlated with the increase in the levels of phosphorylated extracellular signal-regulated kinases (pERK1/2) revealing a potential role of ERK in the HFD mediated liver damage. However APPH treatment reduced the effect of HFD on the apoptosis and fibrosis markers considerably and provided hepato-protection. CONCLUSION: APPH can therefore be considered as an efficient therapeutic agent to ameliorate high fat diet related liver damages.

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPß-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPß, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPß plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

A high-fat diet rich in corn oil induces cardiac fibrosis in rats by activating JAK2/STAT3 and subsequent activation of ANG II/TGF-1ß/Smad3 pathway: The role of ROS and IL-6 trans-signaling.

This study compared the effect of low-fat diet (LFD) and high-fat diet rich in corn oil (HFD-CO) on left ventricular (LV) fibrosis in rats and examined their effect of angiotensin II (ANG II), JAK/STAT, and TGF-1ß/smad3 pathways. As compared to LFD which didn't affect any of the measured parameters, HFD-CO-induced type 2 diabetes phenotype and increased LV collagen synthesis. Mechanistically, it increased LV levels of ROS, ANG II, ACE, IL-6, s-IL-6Rα, TGF-ß1, Smad-3, and activities of JAK1/2 and STAT1/3. AG490, a JAK2 inhibitor, partially ameliorated these effect while Losartan, an AT1 inhibitor completely abolished collagen synthesis. However, with both treatments, levels of ANG II, IL-6, and s-IL-6Rα, and activity of JAK1/STAT3 remained high, all of which were normalized by co-administration of NAC or IL-6 neutralizing antibody. In conclusion: HFD-CO enhances LV collage synthesis by activation of JAK1/STAT3/ANG II/TGF-1ß/smad3 pathway. PRACTICAL APPLICATIONS: We report that chronic consumption of a high-fat diet rich in corn oil (HFD-CO) induces diabetes mellitus phenotype 2 associated with left ventricular (LV) cardiac fibrosis in rats. The findings of this study show that HFD-CO, and through the increasing generation of ROS and IL-6 levels and shedding, could activate LV JAK1/2-STAT1/3  and  renin-angiotensin system (RAS) signaling pathways, thus creating a positive feedback between the two which ultimately leads to activation of TGF-1ß/Smad3 fibrotic pathway. Herein, we also report a beneficial effect of the antioxidant, NAC, or IL-6 neutralizing antibody in preventing such adverse effects of such HFD-CO. However, this presents a warning message to the current sudden increase in idiopathic cardiac disorders, especially with the big shift in our diets toward n-6 PUFA.

Lipidomic and transcriptomic analysis of western diet-induced nonalcoholic steatohepatitis (NASH) in female Ldlr -/- mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, particularly in obese and type 2 diabetic individuals. NAFLD ranges in severity from benign steatosis to nonalcoholic steatohepatitis (NASH); and NASH can progress to cirrhosis, primary hepatocellular carcinoma (HCC) and liver failure. As such, NAFLD has emerged as a major public health concern. Herein, we used a lipidomic and transcriptomic approach to identify lipid markers associated with western diet (WD) induced NASH in female mice. METHODS: Female mice (low-density lipoprotein receptor null (Ldlr -/-) were fed a reference or WD diet for 38 and 46 weeks. Transcriptomic and lipidomic approaches, coupled with statistical analyses, were used to identify associations between major NASH markers and transcriptomic & lipidomic markers. RESULTS: The WD induced all major hallmarks of NASH in female Ldlr -/- mice, including steatosis (SFA, MUFA, MUFA-containing di- and triacylglycerols), inflammation (TNFα), oxidative stress (Ncf2), and fibrosis (Col1A). The WD also increased transcripts associated with membrane remodeling (LpCat), apoptosis & autophagy (Casp1, CtsS), hedgehog (Taz) & notch signaling (Hey1), epithelial-mesenchymal transition (S1004A) and cancer (Gpc3). WD feeding, however, suppressed the expression of the hedgehog inhibitory protein (Hhip), and enzymes involved in triglyceride catabolism (Tgh/Ces3, Ces1g), as well as the hepatic abundance of C18-22 PUFA-containing phosphoglycerolipids (GpCho, GpEtn, GpSer, GpIns). WD feeding also increased hepatic cyclooxygenase (Cox1 & 2) expression and pro-inflammatory ω6 PUFA-derived oxylipins (PGE2), as well as lipid markers of oxidative stress (8-iso-PGF2α). The WD suppressed the hepatic abundance of reparative oxylipins (19, 20-DiHDPA) as well as the expression of enzymes involved in fatty epoxide metabolism (Cyp2C, Ephx). CONCLUSION: WD-induced NASH in female Ldlr -/- mice was characterized by a massive increase in hepatic neutral and membrane lipids containing SFA and MUFA and a loss of C18-22 PUFA-containing membrane lipids. Moreover, the WD increased hepatic pro-inflammatory oxylipins and suppressed the hepatic abundance of reparative oxylipins. Such global changes in the type and abundance of hepatic lipids likely contributes to tissue remodeling and NASH severity.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.

Coreopsis tinctoria Nutt ameliorates high glucose-induced renal fibrosis and inflammation via the TGF-ß1/SMADS/AMPK/NF-κB pathways.

BACKGROUND: Coreopsis tinctoria Nutt is an ethnomedicine widely used in Xinjiang, China. It is consumed as a herbal tea by local Uyghur people to treat high blood pressure and diarrhea. Our previous study confirmed that the ethyl acetate extract of Coreopsis tinctoria (AC) had a protective effect on diabetic nephropathy (DN) in an in vivo experiment. Here we aim to elucidate the protective mechanism of AC and marein, the main ingredient in Coreopsis tinctoria on renal fibrosis and inflammation in vitro under high glucose (HG) conditions. METHODS: A HG-induced barrier dysfunction model in rat mesangial cells (HBZY-1) was established. The cells were exposed to AC and marein and/or HG for 24 h. Then, the renal protective effects of AC and marein via transforming growth factor-ß1 (TGF-ß1)/Smads, AMP-activated kinase protein (AMPK), and nuclear factor kappa beta (NF-κB) signaling were assessed. RESULTS: Both AC and marein suppressed rat mesangial cell hyperplasia and significantly attenuated the expression of HG-disrupted fibrotic and inflammatory proteins in HBZY-1 cells. It was also confirmed that AC and marein remarkably attenuated HG-induced renal inflammation and fibrosis by regulating the AMPK, TGF-ß1/Smads, and NF-κB signaling pathways. CONCLUSION: These results indicated that AC and marein may delay the progression of DN, at least in part, by suppressing HG-induced renal inflammation and fibrosis. Marein may be one of the bioactive compounds in AC.

Effect of chrysanthemum extract on myocardial fibrosis in rats with renovascular hypertension.

OBJECTIVE: To investigate the inhibitory effect of chrysanthemum extract on myocardial fibrosis in rats with renovascular hypertension, and explore the possible mechanism underlying this effect. METHODS: Sixty Wistar rats were randomly divided into six groups: sham operation, model, positive control, and low-, medium-, and high-dose Huai chrysanthemum extract groups (ten rats per group). With the exception of the sham operation group, a renal hypertensive model was established in rats using the ""two-kidney, one clip"" method. After 6 weeks, low-, medium-, and high-dose groups were intragastrically administered chrysanthemum extract at 1, 2, or 4 g/kg, respectively, once daily for 4 weeks. The positive control group was administered Kato Pury at 50 mg/kg once daily for 4 weeks, while sham operation and model groups received an equal volume of distilled water once daily for 4 weeks. Blood pressure changes were examined before modeling, 6 weeks after modeling, and after 4 weeks of treatment administration. Ventricular remodeling indexes were measured by high frequency echocardiography after 4 weeks of treatment administration. Pathological changes were observed by hematoxylin and eosin, and Masson's trichrome staining methods. Collagen typeⅠ (Col Ⅰ) and type Ⅲ (Col Ⅲ) expression were examined by enzyme-linked immunosorbent assays. Transforming growth factor-ß1 (TGF-ß1), sma mad 3 (Smad3), Smad7, Ras homolog gene family, member A (RhoA), and Rho-associated protein kinase 1 (ROCK1) protein expression were detected by Western blot. RESULTS: Compared with the model group, chrysanthemum-administered groups and the positive control group showed significant improvement of arterial blood pressure, echocardiography indicators, and degree of myocardial fibrosis (P < 0.05). In addition, these groups exhibited decreased expression of Col Ⅰ, Col Ⅲ, RhoA, ROCK1, TGF-ß1, and Smad3, and increased Smad7 expression. Such improvements were most obvious in the high-dose chrysanthemum extract group (P < 0.05). CONCLUSION: Chrysanthemum extract could effectively reduce myocardial fibrosis in rats with renovascular hypertension by a mechanism that potentially involves inhibition of RhoA/ROCK1 and TGF-ß1/Smad signaling pathways.

Galectin 3 inhibition attenuates renal injury progression in cisplatin-induced nephrotoxicity.

Nephrotoxicity is a major toxic effect in chemotherapy, which constitutes up to 60% of hospitalized acute kidney injury (AKI). Very few treatment options exist to slow the transition from AKI to subsequent chronic kidney diseases (CKD). Here, we demonstrate that galectin-3 (Gal-3), a ß-galactoside binding lectin that plays an important role in kidney fibrosis and renal failure, is one of the key factors for renal injury progression. Ectopic overexpression of Gal-3 significantly decreased the viability of HEK293, simultaneously inducing of cell cycle arrest and apoptosis. However, inhibition of Gal-3, mediated by modified citrus pectin (MCP), predominantly antagonized the pro-apoptotic effects. Mice were pre-treated with normal or 1% MCP-supplemented drinking water 1 week before cisplatin injection. Analyses of serum creatinine and renal tissue damage indicated that MCP-treated mice demonstrated increased renal function and attenuated renal fibrosis after cisplatin-induced injury. MCP-treated mice also demonstrated decreased renal fibrosis and apoptosis, as revealed by masson trichrome staining and Western blot analysis of cleaved caspase-3. Additionally, the protective role of Gal-3 inhibition in the kidney injury was shown to be mediated by protein kinase C α (PKC-α), which promoted cell apoptosis and collagen I synthesis in HEK293 cells. These results demonstrated the potential Gal-3 and PKC-α as therapeutic targets for the treatment of AKI and CKD.

Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle Fibrosis.

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-ß and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-ß-Smad4 signaling pathway.