Mechanistic efficacy assessment of selected novel methanimine derivatives against vincristine induced Neuropathy: In-vivo, Ex-vivo and In-silico correlates.

Vincristine induced peripheral neuropathy (VIPN) is a serious untoward side effect suffered by cancer patients, which still lacks an adequate therapeutic approach. This study examined the alleviating potential of novel methanimine derivatives i.e. (E)-N-(4-nitrobenzylidene)-4-chloro-2-iodobenzamine (KB 9) and (E)-N-(2-methylbenzylidene)-4-chloro-2-iodobenzamine (KB 10) in VIPN. Vincristine was injected in BALB/c mice for 10 days to instigate nociceptive neuropathy. Dynamic and static allodynia, thermal (hot and cold) hyperalgesia were evaluated at 0, 5, 10 and 14 days using cotton brush, Von Frey filament application, hot plate test, acetone drop and cold water respectively. Tumour necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), lipid peroxide (LPO), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and reactive oxygen species (ROS) assays were performed to assess the efficacy of KB9 and KB10 against neuroinflammation and oxidative stress utilizing ELISA, immunohistochemistry and western blot analysis in brain and sciatic nerve tissues. Computational studies were executed to determine the stable binding conformation of both compounds with respect to COX-2 and NF-κB. Interestingly, both compounds substantially reduced protein expression related to neuroinflammation, oxidative stress (LPO, GST, SOD, CAT) and pain (NF-κB, COX-2, IL-1ß and TNF-α). This molecular analysis suggested that the neuroprotective effect of KB9 and KB10 was mediated via regulation of inflammatory signaling pathways. Overall, this study demonstrated that KB9 and KB10 ameliorated vincristine induced neuropathy, through anti-inflammatory, anti-nociceptive and antioxidant mechanisms.

Ameliorative effect of two structurally divergent hydrazide derivatives against DSS-induced colitis by targeting Nrf2 and NF-κB signaling in mice.

The environmental factors and genetic vulnerability trigger the inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease. Furthermore, the oxidative stress and inflammatory cytokines have been implicated in the aggravation of the IBDs. The aim of the present study was to investigate the effect of N-(benzylidene)-2-((2-hydroxynaphthalen-1-yl)diazenyl)benzohydrazides (NCHDH and NTHDH) compounds against the DSS-induced colitis in mice. The colitis was induced by 5% dextran sulfate sodium (DSS) dissolved in normal saline for 5 days. The effect of the NCHDH and NTHDH on the behavioral, biochemical, histological, and immunohistological parameters was assessed. The NCHDH and NTHDH treatment improved the behavioral parameters such as food intake, disease activity index, and diarrhea score significantly compared to DSS control. The NCHDH and NTHDH treatments significantly increased the antioxidant enzymes, whereas oxidative stress markers were markedly reduced. Similarly, the NCHDH and NTHDH treatments significantly suppressed the activity of nitric oxide (NO), myeloperoxidase (MPO), and eosinophil peroxidase (EPO). The histological studies showed a significant reduction in inflammation, immune cell infiltration, and fibrosis in the NCHDH- and NTHDH-treated groups. The immunohistochemical results demonstrated that NCHDH and NTHDH treatments markedly increase the expression level of Nrf2, HO-1 (hemeoxygenase-1), TRX (thioredoxin reductase), and IκB compared to the DSS-induced group. In the same way, the NCHDH and NTHDH significantly reduced the NF-κB and COX-2 (cyclooxygenase-2) expression levels. The NCHDH and NTHDH treatment significantly improved the symptoms associated with colitis via inducing antioxidants and attenuating oxidative stress markers.

Anti-Inflammatory and Anti-Rheumatic Potential of Selective Plant Compounds by Targeting TLR-4/AP-1 Signaling: A Comprehensive Molecular Docking and Simulation Approaches.

Plants are an important source of drug development and numerous plant derived molecules have been used in clinical practice for the ailment of various diseases. The Toll-like receptor-4 (TLR-4) signaling pathway plays a crucial role in inflammation including rheumatoid arthritis. The TLR-4 binds with pro-inflammatory ligands such as lipopolysaccharide (LPS) to induce the downstream signaling mechanism such as nuclear factor κappa B (NF-κB) and mitogen activated protein kinases (MAPKs). This signaling activation leads to the onset of various diseases including inflammation. In the present study, 22 natural compounds were studied against TLR-4/AP-1 signaling, which is implicated in the inflammatory process using a computational approach. These compounds belong to various classes such as methylxanthine, sesquiterpene lactone, alkaloid, flavone glycosides, lignan, phenolic acid, etc. The compounds exhibited different binding affinities with the TLR-4, JNK, NF-κB, and AP-1 protein due to the formation of multiple hydrophilic and hydrophobic interactions. With TLR-4, rutin had the highest binding energy (-10.4 kcal/mol), poncirin had the highest binding energy (-9.4 kcal/mol) with NF-κB and JNK (-9.5 kcal/mol), respectively, and icariin had the highest binding affinity (-9.1 kcal/mol) with the AP-1 protein. The root means square deviation (RMSD), root mean square fraction (RMSF), and radius of gyration (RoG) for 150 ns were calculated using molecular dynamic simulation (MD simulation) based on rutin's greatest binding energy with TLR-4. The RMSD, RMSF, and RoG were all within acceptable limits in the MD simulation, and the complex remained stable for 150 ns. Furthermore, these compounds were assessed for the potential toxic effect on various organs such as the liver, heart, genotoxicity, and oral maximum toxic dose. Moreover, the blood-brain barrier permeability and intestinal absorption were also predicted using SwissADME software (Lausanne, Switzerland). These compounds exhibited promising physico-chemical as well as drug-likeness properties. Consequently, these selected compounds portray promising anti-inflammatory and drug-likeness properties.

Anti-inflammatory and analgesic potential of leaf extract of Justicia adhatoda L. (Acanthaceae) in Carrageenan and Formalin-induced models by targeting oxidative stress.

Plants are regarded as a valuable and inexpensive source of new drug development, and a variety of plant compounds are now being used in clinical trials to treat a variety of ailments. The goal of this work was to characterize and evaluate the anti-inflammatory and antioxidant effects of Justicia adhatoda L. leaf extract (Acanthaceae). The presence of alkaloids, saponins, tannins, phytosterols, phenols, and proteins in the leaf extract of J. adhatoda was determined using phytochemical screening. While the identification of different compounds in the leaf extract was carried out by HPLC analysis. Similarly, the anti-inflammatory potential of the leaf extract was assessed in Carrageenan and Formalin-induced inflammatory mice models. The phytochemical analysis of the leaf extract indicated a positive test for alkaloids, saponins, tannins, phytosterols, phenols, proteins, and amino acids, while the negative test for carbohydrates, and glycosides, flavonoids, and diterpenes. Moreover, among the detected compounds, gallic acid was found in the highest concentration with a 45.42% composition. The leaf extract showed the highest antibacterial activity against E. coli, while the lowest activity against Listeria was observed. The leaf extract of J. adhatoda revealed promising anti-inflammatory, analgesic, and antioxidants activities both in vitro and in vivo. Similarly, the detected compounds portrayed variable pharmacokinetic as well as binding affinities with the target proteins. In conclusion, the leaf extract exhibited significant antioxidants and antibacterial activities using in vitro assays. Similarly, the extract also revealed promising anti-inflammatory activities in vivo while exhibiting variable Pharmacokinetics and binding affinities towards protein target using computational tools.

N-(benzylidene)-2-((2-hydroxynaphthalen-1-yl)diazenyl)benzohydrazides (1-2) (NCHDH and NTHDH) attenuate DMBA-induced breast cancer via Nrf2/NF-κB/apoptosis signaling.

The present study investigated the effect of the N-(benzylidene)-2-((2-hydroxynaphthalen-1-yl)diazenyl)benzohydrazides (1-2) (NCHDH and NTHDH) against breast cancer using in vitro and in vivo approaches. The NCHDH and NTHDH significantly inhibited the growth of the MCF-7 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The NCHDH and NTHDH treatment significantly inhibited the tumor size, tumor weight, and tumor volume, while it enhanced the survival and tumor free survival rate following 7,12-Dimethylbenz[a]anthracene (DMBA)-induced breast cancer. The NCHDH and NTHDH markedly attenuated the oxidative stress markers and induced the antioxidant level. The enzyme-linked immunosorbent assay (ELISA) showed significant reduction in the inflammatory cytokines production compared with the DMBA control. The NCHDH and NTHDH treatment significantly improved the histological features using hematoxylin and eosin (H and E) staining, Masson's trichrome, PAS (periodic acid Schiff), and Toluidine blue staining compared with the DMBA-induced group. The NCHDH and NTHDH treatment improved the hematological and serological parameters following DMBA-induced breast tumor compared with DMBA-induced group. Furthermore, the NCHDH and NTHDH treatment significantly enhanced the antioxidants signaling proteins such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Heme oxygenase 1 (HO-1). The NCHDH and NTHDH enhanced the inhibitor of NF-κB (IκB) level, while it attenuated the NF-κB level. Similarly, the NCHDH and NTHDH showed marked increase in the apoptosis proteins such as Caspase-3, Caspase-9, and Bcl-2 Associated X-protein (Bax), while it inhibited the B-cell lymphoma 2 (Bcl-2) expression. In conclusion, the NCHDH and NTHDH significantly improved the DMBA-induced breast cancer via attenuating oxidative stress and inflammatory cytokines.

Comprehensive in vivo and in silico approaches to explore the hepatoprotective activity of poncirin against paracetamol toxicity.

In the present study, poncirin was evaluated against paracetamol-induced liver injury using in vivo and computational approaches. Paracetamol was administered intraperitoneally (i.p,) to establish liver injury in mice and, subsequently, to investigate the hepatoprotective effect of poncirin (administered intraperitoneally) on liver injury. The effect of poncirin was evaluated against the liver injury markers and inflammatory cytokines. Similarly, in the present study, the antioxidants and oxidative stress parameters were also assessed following paracetamol-induced liver injury. The histological studies following liver injury were also assessed using H and E staining, Masson's trichrome staining, and periodic acid-Schiff staining. Similarly, the computational approach was used to assess the pharmacokinetic parameters of poncirin and its interaction with various protein targets. Poncirin markedly improved the antioxidant enzymes while attenuated the oxidative stress markers and inflammatory cytokines. Poncirin also markedly improved hematological parameters. Furthermore, poncirin treatment significantly improved the histological parameters using H and E staining, Masson's trichrome, and PAS staining compared to the control. Poncirin treatment also improved the liver function tests and liver synthetic activity compared to paracetamol treated group. The immunohistochemistry analysis revealed significant decrease in the inflammatory signaling protein such as nuclear factor kappa light chain enhancer of activated B cells (NF-κB), Jun N-terminal kinase (JNK), and cyclooxygenase-2 (COX-2) expression level compared to the paracetamol treated group. Computational analysis (molecular docking and molecular dynamic simulation) showed significant binding affinity of poncirin with the NF-κB, JNK, COX-2, IL-1ß, IL-6, and TNF-α via multiple hydrophilic and hydrophobic binds. Similarly, the SwissADME software revealed that poncirin follows various drug-likeness rules and exhibited better pharmacokinetic parameters. Poncirin improved the sign and symptoms associated with liver injury using both in vivo and computational approaches.

Anti-allergic activities of Umbelliferone against histamine- and Picryl chloride-induced ear edema by targeting Nrf2/iNOS signaling in mice.

BACKGROUND: The current study was aimed to investigate the anti-allergic activities of the Umbelliferone (UMB) against the acute Histamine and chronic Picryl chloride (PiCl)-induced allergy in mice. UMB is a coumarin derivative (isolated from Angelica decursiva) found in various parts of the plants such as flowers, roots and, stems isolated from the plants of Umbelliferae family. METHODS: The UMB (1, 10, 50 mg/kg) was administered intraperitoneally (i.p) half an h before or 2 h after the induction of allergic ear edema. The acute ear edema was induced by histamine (intradermally, i.d), while the chronic ear edema was induced by painting the PiCl (sensitized with the toluene) on the ear. The antioxidants and oxidative stress markers were assessed. The histological changes were assessed using Hematoxylin and eosin (H and E) and giemsa staining. The immunohistochemistry studies were performed to assess the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and inducible nitric oxide synthase (iNOS). The data was analyzed using one-way ANOVA tests followed by Tukey's test with p < 0.05 was chosen as criteria for statistical significance. RESULTS: UMB treatment markedly reduced the allergic ear edema and ear weight compared to the negative control. Furthermore, the UMB attenuated the oxidative stress markers, while induced the antioxidants enzymes. Similarly, the UMB treatment significantly attenuated the serum immunoglobulin E (IgE) level. The UMB treatment markedly improved the histological parameters using H and E staining and Giemsa staining. The UMB administration induced the Nrf2 expression, while attenuated the iNOS expression. Furthermore, the computational analysis was performed to assess the interaction of the UMB with the various protein targets and to determine the mechanism of interaction with the target proteins. CONCLUSION: In conclusion, the UMB treatment significantly alleviated the allergic symptoms, attenuating the oxidative stress, improved the histological features using in vivo and computational approaches.

Attenuation of LPS-induced acute lung injury by continentalic acid in rodents through inhibition of inflammatory mediators correlates with increased Nrf2 protein expression.

BACKGROUND: Acute lung injury (ALI) together with acute respiratory distress syndrome (ARDS) are associated with high rate of mortality and morbidity in patients. In the current study, the anti-inflammatory effects of continentalic acid (CNT) in LPS-induced acute lung injury model was explored. METHODS: The acute lung injury model was established by administering LPS (5 mg/kg) intraperitonealy. Following LPS administration, the survival rate, temperature changes and lung Wet/Dry ratio were assessed. The antioxidants (GSH, GST, Catalase and SOD) and oxidative stress markers (MDA, NO, MPO) were evaluated in all the treated groups. Similarly, the cytokines such as IL-1ß, IL-6 and TNF-α were analyzed using ELISA assay. The histological changes were determined using H and E staining, while Nrf2 and iNOS level were determined using immunohistochemistry analysis. The molecular docking analysis was performed to assess the pharmacokinetics parameters and interaction of the CNT with various protein targets. RESULTS: The results showed that CNT dose dependently (10, 50 and 100 mg/kg) reduced mortality rate, body temperature and lungs Wet/Dry ratio. CNT post-treatment significantly inhibited LPS-induced production of pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. The CNT post-treatment markedly improved the hematological parameters, while significantly reduced the MPO (indicator of the neutrophilic infiltration) activity compared to the LPS treated group. Furthermore, the CNT (100 mg/kg) post-administration remarkably inhibited the lung Wet/Dry ratio. The CNT (100 mg/kg) treated group showed marked reduction in the oxidative stress markers such as malonaldehyde (MDA) and Nitric oxide (NO) concentration, while induced the level of the anti-oxidant enzymes such as GST, GSH, Catalase and SOD. Similarly, the CNT markedly reduced the iNOS expression level, while induced the Nrf2 protein expression. Additionally, the molecular docking study showed significant binding interaction with the Nrf2, p65, Keap1, HO-1, IL-1ß, IL-6, TNF-α and COX-2, while exhibited excellent physicochemical properties. CONCLUSION: The CNT showed marked protection against the LPS-induced lung injury and improved the behavioral, biochemical and histological parameters. Furthermore, the CNT showed significant interaction with several protein targets and exhibited better physicochemical properties.

Poncirin attenuates CCL4-induced liver injury through inhibition of oxidative stress and inflammatory cytokines in mice.

BACKGROUND: In the present study, the poncirin which is flavonoid-7-o-glycosides (isolated from the Poncirus trifoliata) in nature was evaluated against the Carbon tetra chloride (CCL4)-induced liver injury. The poncirin have been reported for various anti-inflammatory, analgesic activity etc. Based on the previous studies it was anticipated that the poncirin will ameliorate CCL4-induced liver injury. METHODS: The CCL4-induced acute and chronic liver injury model (albino BALB/c mice) was used. Following the induction of the liver injury various parameters such as food and water intake, body weight and weight to dry ratio changes were assessed. Furthermore, various hematological, biochemical parameters and histological studies such as hemotoxylin and eosin (H and E) staining were performed. The poncirin treatment was also evaluated against the pro-inflammatory cytokines such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) using enzyme link immunosorbant assay (ELISA). The Swiss Target prediction software was used to investigate interaction of the poncirin on the various hepatic enzymes. RESULTS: The poncirin treatment markedly improved the behavioral parameters such as food and water intake. The liver weight variation was attenuated and total body was improved markedly. The hematological and biochemical parameters were significantly improved compared to the CCL4 treated groups. The anti-oxidants were induced, while oxidative stress markers were reduced promisingly. The H and E staining showed that poncirin treatment significantly improved the histology of liver compared to the CCL4 treated group. Furthermore, the poncirin treatment also evidently decreased the inflammatory mediators. CONCLUSIONS: The poncirin treatment showed marked improvement in behavioral, biochemical and histological parameters following CCL4-induced liver injury. Additionally, the poncirin treatment also markedly improved the antioxidant enzymes, attenuated the oxidative stress markers and inflammatory cytokines.

Anomalin attenuates LPS-induced acute lungs injury through inhibition of AP-1 signaling.

In the present study, the anomalin was investigated to determine the protective effects and underlying mechanism against LPS-induced acute lung injury in mice. Anomalin administration 30 min after the LPS injection, significantly attenuated the mechanical allodynia, decrease body temperature, and improved the histological changes and inhibited the infiltration of leukocytes. The anomalin treatment markedly inhibited the production of pro-inflammatory mediators such as cytokines (IL-1ß, IL-6 and TNF-α) and NO in contrast to the LPS treated groups. Similarly, the anomalin also enhanced the level of anti-oxidant enzymes such as GST, GSH, Catalase and inhibited oxidative stress marker such as MDA. In order to explore the molecular mechanism the effect of anomalin was evaluated for mitogen activated protein kinases (MAPK) in LPS-stimulated RAW264.7 cells. The anomalin treatment significantly attenuated the MAPK proteins such as ERK1/2, JNK and p38 (which is downstream signaling proteins to the MAPKKKs and MAPKKs protein) in the RAW264.7 macrophages using western blot analysis. Furthermore, the western blot analysis showed that anomalin treatment significantly inhibited the activation of the Akt proteins in the RAW264.7 macrophages. The AP-1 served as downstream target for the MAPK pathways and the blocking MAPK pathways is responsible for the inhibition of the AP-1 protein. The AP-1/DNA binding was assessed in the RAW264.7 cells using EMSA. The anomalin treatment significantly restricted the AP-1/DNA binding activity and the decrease in the AP-1/DNA binding activity might be contributed due to the upstream inhibition of the MAPKs signaling.

Antinociceptive properties of 25-methoxy hispidol A, a triterpinoid isolated from Poncirus trifoliata (Rutaceae) through inhibition of NF-κB signalling in mice.

The 25-methoxy hispidol A (25-MHA) is a triterpenoid, isolated from the immature fruit of Poncirus trifoliata (Rutaceae). The pretreatment with 25-MHA markedly (p < 0.001) attenuated the formalin-induced biphasic responses as well as acetic acid-induced writhing responses. The intraperitoneal administration of 25-MHA significantly attenuated the mechanical hyperalgesia (p < 0.001) and allodynia (p < 0.05). Similarly, 25-MHA also significantly attenuated (p < 0.001) complete Freund's adjuvant (CFA)-induced paw edema in mice. The 25-MHA treatment significantly attenuated the production of nuclear kappa B (NF-κB) (p65 nuclear subunit). The cytokines are the important mediators of inflammation and pain; however, treatment with 25-MHA exhibited significant inhibition (p < 0.001) on the mRNA expression levels of various inflammatory mediators. The 25-MHA administration also significantly enhanced antioxidant enzymes (p < 0.001) and inhibited the oxidative stress markers. The current study indicates that 25-MHA significantly (p < 0.001) inhibited the nitric oxide (NO) in mice plasma. Similarly, the haematoxylin and eosin (H&E) staining shows that 25-MHA administration significantly inhibited the inflammatory process in the mice paw tissue compared with the CFA-treated group. The 25-MHA treatment did not exhibited any toxicity on the liver, kidney, muscles strength, and motor co-ordination in mice. The 25-MHA was coadministered with the various drugs such as tramadol, piroxicam, and gabapentin to observe the synergistic effect.