Manjari Medika Grape Seed Extract Protects Methotrexate-Induced Hepatic Inflammation: Involvement of NF-κB/NLRP3 and Nrf2/HO-1 Signaling System

Abstract

Objective

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

Abbreviations

AICR TFase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; ALP, Alkaline phosphatase; ALT, Alanine transaminases; AST, Aspartate transaminases; BODIPY, 5,5-Difluoro-5H-4λ⁵-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ylium-5-uide; BSA, Bovine serum albumin; CaCl₂, Calcium chloride; COX-2, cyclooxygenase-2; DAF-FM DA, 4-amino-5-(methylamino)-2′,7′-difluorofluorescein diacetate; DAPI, 4′,6-diamidino-2-phenylindole; DCF, Dichlorofluorescein; DMEM, Dulbecco’s Modified Eagle Medium; FBS, Fetal bovine serum; FITC, Fluorescein isothiocyanates; GdCl₃, gadolinium chloride; H₂DCFDA, 2′,7′-dichlorofluorescein diacetate; HBSS, Hanks’ Balanced Salt Solution; HO-1, Heme oxygenase-1; IgG, Immunoglobulin G; IL-1β, Interleukin 1 beta; iNOS, inducible nitric oxide synthase; LC-MS, Liquid chromatography–mass spectrometry; LDH, Lactate dehydrogenase; MAPKs, Mitogen-activated protein kinases; NAC, N-Acetylcysteine; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NLR family pyrin domain containing 3; NO, Nitric oxide; Nrf2, nuclear factor erythroid 2–related factor 2; PBS, Phosphate buffer saline; PE, Phycoerythrin; RNS, Reactive nitrogen species; SOD, Super oxide dismutase; TGF-β, Transforming growth factor-β; TLR4, Toll-like receptor 4; Tris-HCl, Tris(hydroxymethyl)aminomethane hydrochloride; UPLC-(ESI)-QToF-MS, Ultra performance liquid chromatography-Electro spray ionization-Quadrupole Time-of-Flight-Mass spectrometry; α-1AT, Alpha-1 antitrypsin; α-SMA, Alpha-Smooth Muscle Actin.

Ethics Approval and Informed Consent

All experimental protocols were performed as per the guidelines of the Institutional Animal Ethics Committee, CSIR-Indian Institute of Chemical Biology and approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (Ministry of Environment, Forests and Climate Change, The Government of India) (Approval No. IICB/AEC/Meeting/Feb/2018/1).

Acknowledgments

We are grateful to Debalina Chakraborty, Binayak Pal and Banasri Das of Central Instrumentation Facility, CSIR-Indian Institute of Chemical Biology for providing flow cytometer and confocal microscope facilities. Equally, we express our thankfulness to Suparna Banerjee for improving the language of the manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Kaushik Banerjee and Krishna Das Saha are co-correspondence authors.

Disclosure

The authors declare no conflicts of interest in relation to this work.

Additional information

Funding

This work is supported by the National Medicinal Plant Board (Ministry of AYUSH, Government of India).