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Krishnendu Manna,1,* Zareen S Khan,2,* Moumita Saha,3,* Snehasis Mishra,3 Nilesh Gaikwad,4 Jatindra Nath Bhakta,1 Kaushik Banerjee,2 Krishna Das Saha3 1Department of Food & Nutrition, University of Kalyani, Nadia, West Bengal, India; 2National Referral Laboratory, ICAR-National Research Centre for Grapes, Pune, Maharashtra, 412307, India; 3Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, 700032, India; 4ICAR-National Research Centre on Pomegranate, Solapur, Maharashtra, 413255, India*These authors contributed equally to this workCorrespondence: Krishna Das Saha, Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, West Bengal, 700032, India, Tel +91 33 2499 5810, Fax +91 33 2473 5197, Email krishna@iicb.res.in Kaushik Banerjee, National Referral Laboratory, ICAR-National Research Centre for Grapes, Pune, Maharashtra, 412307, India, Email kbgrape@yahoo.comObjective: Grape Seed Extract is a natural source of various polyphenols, which have been shown to possess potent antioxidant and free radical-scavenging activities. The earlier studies have reported that grape seed extract exhibits broad-spectrum pharmacological activities. Therefore, studying the hepatoprotective effects and elucidation of mechanisms of action of the Indian Variety, Manjari Medika grape seed extract (GSE), may give an insight into therapeutic benefits. Methotrexate (MTX) is the first-line pharmacological therapy for different rheumatic diseases. The major adverse events such as hepatotoxicity are evident even in the low doses used for the treatment. The present study investigated the role of MTX on hepatic damage in murine liver and the plausible protective effects of the Indian grape variety, Manjari Medika grape seed extract, in ameliorating it.Methods and Results: To assess the hepatological modulation, mice were divided into eight groups to investigate the ameliorative potential of this GSE (75 and 125 mg/kg) and correlate the experimental findings. The active components of the extract were assessed through UPLC-(ESI)-QToF-MS analysis. On the other hand, various biochemical and immunological indices were carried out to correlate the experimental data. The result demonstrated that the prophylactic administration of GSE reduced MTX-induced hepatic toxicity indices, which subsequently restored the hepatic morphological architecture. Moreover, the application of GSE in a dual dosage (75 and 125 mg/kg) suppressed MTX-induced reactive oxygen species generation, followed by lipid peroxidation and cellular nitrite formation. MTX-induced inflammasome activation through the redox-assisted cascade of TLR4/NF-κB signaling was further reduced by applying the GSE. The results showed that the activation of cytoprotective transcription factor Nrf2 enhanced the level of endogenous antioxidants. Furthermore, through the regulation of TLR4/NF-κB and Nrf2/HO-1 axis, this extract could reduce the MTX-mediated hepatic damage.Conclusion: Our findings suggest that Manjari Medika seed extract could be used as a therapeutic agent to relieve the side effects of MTX and other hepatic disorders.Graphical Abstract: Keywords: hepatic fibrosis, Kupffer cells, Manjari Medika grape seed extract, proanthocyanidins, inflammasome, antioxidant response

Moumita Saha,1 Krishnendu Manna,2 Krishna Das Saha1 1Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India; 2Department of Food and Nutrition, University of Kalyani, Kalyani, West Bengal, IndiaCorrespondence: Krishna Das Saha, Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata, West Bengal, 700032, India, Tel +91 33 2499 5810, Fax +91 33 2473 5197, Email krishna@iicb.res.inBackground: NLRP3 inflammasome activation plays a critical role in mediating inflammation and NASH (non-alcoholic steatohepatitis) progression that ultimately leads to cirrhosis and hepatocellular carcinoma. Melatonin (MLT) controls high-fat diet-induced NASH in the murine model by modulating NLRP3 mediated inflammation. P2X7R-mediated inflammasome activation is reported in several inflammatory models including NASH.Objective: The role of MLT in P2X7R-mediated inflammation in the NASH model has not yet been explored. The present study investigated the role of MLT in amending high-fat diet-induced nonalcoholic steatohepatitis in the murine liver.Methods: To evaluate the hepatological changes, mice were divided into four groups to investigate the improvement potential of this MLT (10 and 20 mg/kg) and to assess the experimental findings. Histology, biochemical assays, ELISA, FACS analysis, Western blotting, and IF were performed to assess the physical and molecular changes upon melatonin treatment.Results: The result demonstrated that MLT administration reduced HFD (high-fat diet)-induced non-alcoholic steatohepatitic indices, which successively restored the hepatic morphological architecture and other pathophysiological features too. Moreover, the application of MLT suppressed HFD-induced activation of the inflammasome and through TLR4/NF-κB signaling. Herein, we report that MLT significantly suppresses P2X7R expression and calcium influx along with inflammasome in both in vitro and in vivo. The docking study revealed a strong binding affinity of MLT with P2X7R. Moreover, the results also showed that the Nrf2 level was boosted which may normalize the expression of antioxidant proteins that safeguard against oxidative damage triggered by inflammation. Furthermore, some matrix metalloproteinases like MMP 2 and MMP 9 were repressed and TIMP-1 level was increased, which also signifies that MLT could improve liver fibrosis in this model.Conclusion: Based on our findings, this study may conclude that MLT could be used as a therapeutic agent in the high-fat diet-induced NASH model as it has persuasive anti-inflammatory potential.Graphical Abstract: Keywords: melatonin, NASH, NAFLD, inflammasome, P2X7, inflammation, Kupffer cells