Anti-inflammatory and Anti-cancer Mechanisms of (E)-Nꞌ-(4-Chlorobenzylidene)-2-(E-(2- hydroxynaphthalen-1-yl)diazenyl)benzohydrazide (NCHDH) and (E)-Nꞌ-(3,4,5-trimethoxybenzylidene)- 2-(E-(2-hydroxynaphthalen-1- yl)diazenyl)benzohydrazide (NTHDH)

Abstract

The present study investigated the anti-inflammatory and anti-cancer activities of the (E)-Nꞌ-(4-Chlorobenzylidene)-2-(E-(2-hydroxynaphthalen-1- yl)diazenyl)benzohydrazide (NCHDH) and (E)-Nꞌ-(3,4,5-trimethoxybenzylidene)-2- (E-(2-hydroxynaphthalen-1-yl) diazenyl)benzohydrazide (NTHDH). The NCHDH and NTHDH were investigated against the LPS-induced sepsis in rodents. The NCHDH and NTHDH markedly improved the physiological sign and symptoms associated with the sepsis such as mortality, temperature, and clinical scoring compared to control. The NCHDH and NTHDH also inhibited the production of the NO and MPO when compared to LPS-induced. Furthermore, the treatment control improved the histopathology of all the major organs using H and E staining markedly. Additionally, the Masson’s trichrome and PAS (Periodic Acid Schiff) staining also showed improvement in the NCHDH and NTHDH treated group in contrast to LPS-induced group. The antioxidants were enhanced by the intervention of the NCHDH and NTHDH and the level of the MDA and POD were significantly inhibited in all the major organs in contrast to only LPS treated group. The NCHDH and NTHDH markedly enhanced the Nrf2, HO-1 (Heme oxygenase-1), while attenuated the Keap1 and TRPV1 expression level in all the major organs following LPS challenge. Moreover, the NCHDH and NTHDH treatment showed marked increased in the mRNA expression level of the HSP70/90 proteins in contrast to the negative control. Furthermore, the effect of NCHDH and NTHDH was evaluated against the DSS-induced colitis in mice. The DSS administration exhibited evident alteration in the various parameters associated with the colitis such as biochemical, histological, and immunohistological. However, those animals which were treated with the NCHDH and NTHDH showed significant improvement in the various parameters such as survival rate, food consumption, disease activity index and diarrhea score. The NCHDH and NTHDH treatment also significantly increased the antioxidant enzymes, while attenuated the oxidative stress markers. The treatment control showed notable increase in the expression level of the Nrf2 and HO-1 proteins, while attenuated the Keap1 expression. Similarly, the treatment control also showed marked reduction in the NF-κB and COX 2 (cyclooxygenase-2) level in contrast to DSS control. Additionally, the NCHDH and NTHDH were evaluated against the acute Carrageenan and chronic CFA-induced models. The NCHDH and NTHDH treatment markedly attenuated the inflammatory xi and analgesic parameters in contrast to CFA treated group. Furthermore, the increase in the oxidative stress and attenuation of antioxidant enzymes has been reported following CFA administration. However, NCHDH and NTHDH treatment significantly induced the antioxidants and attenuated the oxidative stress markers. The Western blot, immunohistology and ELISA assay revealed marked increase in the NF-κB level, Jun N-terminal Kinase (JNK) level, TNF-α, and COX-2 level, however, the NCHDH and NTHDH attenuated their expressions significantly. Similarly, the NCHDH and NTHDH significantly induced the mRNA expression levels of heat shock proteins. The NCHDH and NTHDH were evaluated against the DMBA-induced mammary tumor in mice. The NCHDH and NTHDH treatment significantly inhibited the tumor size, tumor weight, tumor volume, while enhanced the survival and tumor free survival rate. The NCHDH and NTHDH significantly inhibited the oxidative stress markers, inflammatory cytokines, while enhanced the antioxidants level. The NCHDH and NTHDH treatment significantly altered the histological features following staining with the H and E, Masson trichrome, PAS, and Toluidine Blue compared to the DMBA induced group. Furthermore, the NCHDH and NTHDH treatment significantly enhanced the antioxidants enzymes such as Nrf2, HO-1, while inhibited the expression of the NF-κB signaling using immunohistochemistry analysis. Furthermore, the NCHDH and NTHDH was evaluated against the in vitro and in vivo DMH-induced colon cancer in mice. The NCHDH and NTHDH treatment significantly increase the survival, incidence of cancer, and other behavioral parameters. Similarly, the DMH induced group also showed marked decrease in the antioxidants level, while enhanced the oxidative stress and inflammatory mediators. However, the NCHDH and NTHDH treatment showed increase in the antioxidants, while attenuated the oxidative stress. Similarly, the NCHDH and NTHDH showed marked decrease in the NF-κB, IκB, Bcl 2, while enhanced the expression of the Caspase-3, Caspase-9, Bax, Nrf2 and HO-1. Key words: Inflammation, Cancer, sepsis, arthritis, colitis, cytokines, NCHDH and NTHDH