Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Thyroid Hormone Decreases Hepatic Steatosis, Inflammation, and Fibrosis in a Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, lobular inflammation, and fibrosis. Thyroid hormone (TH) reduces steatosis; however, the therapeutic effect of TH on NASH-associated inflammation and fibrosis is not known. This study examined the therapeutic effect of TH on hepatic inflammation and fibrosis during NASH and investigated THs molecular actions on autophagy and mitochondrial biogenesis. Methods: HepG2-TRß cells were treated with bovine serum albumin-conjugated palmitic acid (PA) to mimic lipotoxic conditions in vitro. Mice with NASH were established by feeding C57BL/6J mice Western diet with 15% fructose in drinking water for 16 weeks. These mice were administered triiodothyronine (T3)/thyroxine (T4) supplemented in drinking water for the next eight weeks. Results: In cultured HepG2-TRß cells, TH treatment increased mitochondrial respiration and fatty acid oxidation under basal and PA-treated conditions, as well as decreased lipopolysaccharides and PA-stimulated inflammatory and fibrotic responses. In a dietary mouse model of NASH, TH administration decreased hepatic triglyceride content (3.19 ± 0.68 vs. 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 vs. 2.58 ± 0.30 mg/g liver) when compared with mice with untreated NASH. Metabolomics profiling of lipid metabolites showed that mice with NASH had increased triacylglycerol, diacylglycerol, monoacylglycerol, and hepatic cholesterol esters species, and these lipid species were decreased by TH treatment. Mice with NASH also showed decreased autophagic degradation as evidenced by decreased transcription Factor EB and lysosomal protease expression, and accumulation of LC3B-II and p62. TH treatment restored the level of lysosomal proteins and resolved the accumulation of LC3B-II and p62. Impaired mitochondrial biogenesis was also restored by TH. The simultaneous restoration of autophagy and mitochondrial biogenesis by TH increased ß-oxidation of fatty acids. Additionally, the elevated oxidative stress and inflammasome activation in NASH liver were also decreased by TH. Conclusions: In a mouse model of NASH, TH restored autophagy and mitochondrial biogenesis to increase ß-oxidation of fatty acids and to reduce lipotoxicity, oxidative stress, hepatic inflammation, and fibrosis. Activating thyroid hormone receptor in the liver may represent an effective strategy for NASH treatment.

Hyperhomocysteinemia causes ER stress and impaired autophagy that is reversed by Vitamin B supplementation.

Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.

Abrogation of nimesulide induced oxidative stress and mitochondria mediated apoptosis by Fumaria parviflora Lam. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Fumaria parviflora Lam. is used for treating aches and pains, diarrhea, fever, influenza and other complications. The herb mixed with honey is taken to prevent vomiting as per Ayurvedic text. AIM OF THE STUDY: In vivo studies were conducted to explore the hepatoprotective potential of Fumaria parviflora Lam. Fp extract against nimesulide induced oxidative stress and regulation of critical events in mitochondria mediated apoptosis. MATERIALS AND METHODS: Group of Wistar rats were fed with nimesulide for 5 days (80 mg/kg/day, po), another group was pre-treated with Fp extract/silymarin (200mg/kg/day, po) for 5 days followed by nimesulide exposure. Liver serum biomarkers and histopathology were done to assess hepatotoxicity caused by nimesulide. Antioxidant enzymes (SOD, LPO, GPx, GR) were assessed using biochemical assays as well as gene expression by RT-PCR. GSH content and ROS generation was also evaluated using flow cytometry. Key apoptotic markers like phosphatidyl serine externalization, Bax, Bcl-2 translocation, mitochondrial membrane potential, cytochrome c release, caspases (9/3) activation and DNA damage were also observed in all the groups to confirm involvement of mitochondrial pathway. RESULTS: Pre-treatment with Fp extract for 5 days significantly reduced the impact of nimesulide induced toxicity as evident from the serum biomarkers of liver damage and histopathology. It also modulated antioxidant enzymes mRNA expression as well as activity (SOD, glutathione peroxidase, glutathione reductase) and reduced lipid peroxidation during nimesulide toxicity. Nimesulide exposure decreased GSH content (92.9%) and increased reactive oxygen species (9.29 fold) which was attenuated in Fp treated rats. Fp pre-treatment significantly altered key apoptotic events like Bcl2 and Bax translocation, inhibited mitochondrial depolarization, prevented cytochrome c release, caspase-9/caspase-3 activation and DNA damage. CONCLUSION: Our in vivo findings regarding protection accorded by Fp extract against nimesulide toxicity suggest that Fp not only reduced hepatotoxicity but attenuated critical control points of apoptotic cell death.

Involvement of mitochondria mediated pathways in hepatoprotection conferred by Fumaria parviflora Lam. extract against nimesulide induced apoptosis in vitro.

Nimesulide, a popular nonsteroidal anti-inflammatory drug, has been associated with serious hepatotoxicity. Reactive oxygen species (ROS) and mitochondrial perturbations have been implicated in drug induced hepatotoxicity, although their role in the pathway needs exploration. Study was undertaken to elucidate the effect of Fumaria parviflora Lam. (Fp) on nimesulide induced cell death in primary rat hepatocyte cultures. Fp extract treated cells showed increased viability as compared to nimesulide stressed cells as assessed by MTT assay. LDH leakage increased significantly at 500microM nimesulide, and the data suggested that apoptosis was the predominant mechanism responsible for cell death. Nimesulide induced apoptosis was further confirmed by DNA fragmentation and chromatin condensation. Nimesulide exposure increased intracellular ROS, translocation of Bax and Bcl2 followed by mitochondrial depolarization and cytochrome c (Cyt c) release along with caspase-9/-3 activity confirming involvement of mitochondria in nimesulide induced apoptosis. Events like membrane depolarization of mitochondria, expression of Bax, Bcl2, externalization of phosphatidyl serine are substantially reversed by the pre-treatment of Fp extract. Thus, the study indicates that Fp extract modulates critical events regulating pro and anti-apoptotic proteins in mitochondria dependent apoptosis induced by nimesulide.