Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 µg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

Cooperative STAT3-NFkB signaling modulates mitochondrial dysfunction and metabolic profiling in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) continues to pose a significant health challenge and is often diagnosed at advanced stages. Metabolic reprogramming is a hallmark of many cancer types, including HCC and it involves alterations in various metabolic or nutrient-sensing pathways within liver cells to facilitate the rapid growth and progression of tumours. However, the role of STAT3-NFκB in metabolic reprogramming is still not clear. APPROACH AND RESULTS: Diethylnitrosamine (DEN) administered animals showed decreased body weight and elevated level of serum enzymes. Also, Transmission electron microscopy (TEM) analysis revealed ultrastructural alterations. Increased phosphorylated signal transducer and activator of transcription-3 (p-STAT3), phosphorylated nuclear factor kappa B (p-NFκß), dynamin related protein 1 (Drp-1) and alpha-fetoprotein (AFP) expression enhance the carcinogenicity as revealed in immunohistochemistry (IHC). The enzyme-linked immunosorbent assay (ELISA) concentration of IL-6 was found to be elevated in time dependent manner both in blood serum and liver tissue. Moreover, immunoblot analysis showed increased level of p-STAT3, p-NFκß and IL-6 stimulated the upregulation of mitophagy proteins such as Drp-1, Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Meanwhile, downregulation of Poly [ADP-ribose] polymerase 1 (PARP-1) and cleaved caspase 3 suppresses apoptosis and enhanced expression of AFP supports tumorigenesis. The mRNA level of STAT3 and Drp-1 was also found to be significantly increased. Furthermore, we performed high-field 800 MHz Nuclear Magnetic Resonance (NMR) based tissue and serum metabolomics analysis to identify metabolic signatures associated with the progression of liver cancer. The metabolomics findings revealed aberrant metabolic alterations in liver tissue and serum of 75th and 105th days of intervention groups in comparison to control, 15th and 45th days of intervention groups. Tissue metabolomics analysis revealed the accumulation of succinate in the liver tissue samples, whereas, serum metabolomics analysis revealed significantly decreased circulatory levels of ketone bodies (such as 3-hydroxybutyrate, acetate, acetone, etc.) and membrane metabolites suggesting activated ketolysis in advanced stages of liver cancer. CONCLUSION: STAT3-NFκß signaling axis has a significant role in mitochondrial dysfunction and metabolic alterations in the development of HCC.

Mitochondrial-mediated nuclear remodeling and macrophage polarizations: A key switch from liver fibrosis to HCC progression.

Liver fibrosis is a significant health burden worldwide and has emerged as the leading cause of Hepatocellular carcinoma (HCC) incidence. Mitochondria are the dynamic organelles that regulate the differentiation, survival, and polarization of macrophages. Nuclear-DNA-associated proteins, micro-RNAs, as well as macrophage polarization are essential for maintaining intracellular and extra-cellular homeostasis in the liver parenchyma. Dysregulated mitochondrial coding genes (ETS complexes I, II, III, IV, and V), non-coding RNAs (mitomiRs), and nuclear alteration lead to the production of reactive oxygen species (ROS) and inflammation which are implicated in the transition of liver fibrosis into HCC. Recent findings indicated the protecting effect of E74-like factor 3/peroxisome proliferator-activated receptor-γ (Elf-3/PPAR-γ). HDAR-y inhibits the deacetylation of PPAR-y and maintains the PPAR-y pathway. Elf-3 plays a tumor suppressive role through epithelial-mesenchymal transition-related gene and zinc finger E-box binding homeobox 2 (ZEB-2) domain. Additionally, the development of HCC includes the PI3K/Akt/mTOR and transforming Growth Factor ß (TGF-ß) pathway that promotes the Epithelial-mesenchymal transition (EMT) through Smad/Snail/Slug signaling cascade. In contrast, the TLR2/NOX2/autophagy axis promotes M2 polarization in HCC. Thus, a thorough understanding of the mitochondrial and nuclear reciprocal relationship related to macrophage polarization could provide new research opportunities concerning diseases with a significant impact on liver parenchyma towards developing liver fibrosis or liver cancer. Moreover, this knowledge can be used to develop new therapeutic strategies to treat liver diseases.

CYP2E1 triggered GRP78/ATF6/CHOP signaling axis inhibit apoptosis and promotes progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. Cytochrome P450 2E1 (CYP2E1) is an enzyme, primarily involved in the metabolism of xenobiotics and procarcinogens. The present study was designed to investigate the potential role of CYP2E1 triggered endoplasmic reticulum stress in the progression of HCC through inhibition of apoptosis. In vitro CYP2E1 promotes HepG2 cell migration, reduced chromatin condensation, enhanced intracellular ROS accumulation and induce cell cycle progression. Conversely this effect was averted by CYP2E1 siRNA, selective inhibitor Diallyl sulphide (DAS) and antioxidants (vitamin C and E). In vivo Diethylnitrosamine (DEN) induced HCC rats showed decreased body weight and increased relative liver weight. Moreover, macro trabecular-massive HCC (MTM-HCC) histological subtyping showed pathological features like well-differentiated tumors, micro-trabecular and pseudo glandular patterns, megakaryocytes and cholestasis. Masson's trichrome staining revealed an intensive accumulation of collagen fibers in the extracellular matrix (ECM). Increased CYP2E1, VEGF and PCNA enhance the carcinogenicity as revealed in immunohistochemistry results. Immunoblot analysis showed reduced expression of copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) in cytosolic as well as mitochondrial fraction of rat liver tissue respectively. Also, increased level of CYP2E1 stimulated the upregulation of unfolded proteins response (UPR) and ER stress-related proteins such as Glucose regulatory protein 78 (GRP78), activating transcription factor 6 (ATF6) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). Meanwhile, CYP2E1 stimulated ER-stress reduces BCL2 and downregulates the cleaved caspase 3 thus suppresses apoptosis. in. Furthermore, immunofluorescence revealed increased expression level of α-SMA in the HCC rat liver tissue. The level of CYP2E1 mRNA was significantly increased. Altogether, these findings indicate that CYP2E1 has a dynamic role in the pathogenesis of HCC and might be a budding agent in liver carcinogenesis therapy.

CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homolog (CLUH) only in the active UC tissues compared with the unaffected areas from the same patient and healthy controls. Stimulation with bacterial Toll-like receptor (TLR) ligands similarly reduced CLUH expression in human primary macrophages. Further, CLUH negatively regulated secretion of proinflammatory cytokines IL-6 and TNF-α and rendered a proinflammatory niche in TLR ligand-stimulated macrophages. CLUH was further found to bind to mitochondrial fission protein dynamin related protein 1 (DRP1) and regulated DRP1 transcription in human macrophages. In the TLR ligand-stimulated macrophages, absence of CLUH led to enhanced DRP1 availability for mitochondrial fission, and a smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production and reduced mitophagy and lysosomal function in CLUH-knockout macrophages. Remarkably, our studies in the mouse model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report to our knowledge explaining the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

Benzo[a]pyrene treatment modulates Nrf2/Keap1 axis and changes the metabolic profile in rat lung cancer.

Lung cancer is an aggressive malignancy and the leading cause of cancer-related deaths. Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon, plays a pivotal role in lung carcinogenesis. Uncovering the molecular mechanism underlying the pathophysiology of B[a]P induced malignancy is crucial. Male Sprague Dawley rats were induced with B[a]P to generate a lung cancer model. The B[a]P administered rats show increased body and lung weight, loss of normal pulmonary architecture, and decreased survival. This study demonstrated that B[a]P upregulates activating transcription factor-6 (ATF6) and C/EBP Homologous Protein (CHOP) and induces endoplasmic reticulum (ER) stress. B[a]P also dysregulated mitochondrial homeostasis by upregulating, PTEN-induced putative kinase-1 (PINK1) and Parkin. B[a]P affected the levels of superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA), and increased oxidative stress. B[a]P exposure downregulated Kelch-like ECH-associated protein 1 (Keap1) and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and Heme oxygenase-1(HO1). The metabolomic study identified that biosynthesis of nucleotide, amino acids, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), and glutathione metabolism were up-accumulated. On the other hand, lower accumulation of fatty acids e.g., palmitic acid, stearic acid, and oleic acid were reported in the B[a]P induced group. Overall, the results of this study indicate that B[a]P treatment affects several signaling and metabolic pathways, whose dysregulation might be involved in lung cancer induction.

Alternanthera brasiliana L. extract alleviates carbon tetrachloride-induced liver injury and fibrotic changes in mice: Role of matrix metalloproteinases and TGF-ß/Smad axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera brasiliana L. is a flowering plant belonging to the family Amaranthaceae and is popularly known as "penicillin". It is used in folk medicine to treat infections, coughs, wound healing, and inflammatory diseases. AIM OF THE STUDY: We investigated the effect of Alternanthera brasiliana L. leaves hydroalcoholic extract (AB) against oxidative stress, inflammation, and fibrotic changes in an experimental model of carbon tetrachloride (CCl4)-induced liver injury and fibrosis in mice. MATERIALS AND METHODS: Thirty-six male Balb/C mice were randomized into five groups: normal control, AB control, CCl4 control, CCl4 + AB-200 mg/kg, and CCl4 + AB-400 mg/kg. In mice, liver injury was induced by intraperitoneal injection of CCl4 (20% in corn oil, 5 ml/kg body weight) thrice a week for six consecutive weeks. AB extract at two doses (200 mg/kg and 400 mg/kg body weight) was administered orally for six consecutive weeks. Liver injury-related serum markers (ALT, AST, ALP), antioxidants (GSH, GST, SOD, and vitamin C), pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-18, ultrasonographic and histological alterations, proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), nuclear factor-κB (p65) (NF-κB), nod-like receptor protein 3 (NLRP3), and TGF-ß/Smad signaling were accessed. LC-Q-TOF-MS/MS analysis of AB was performed. RESULTS: AB treatment significantly decreased the CCl4-induced rise in serum ALT, AST, and ALP activities and improved the histological alterations. Compared with the CCl4-treated group, treatment with AB significantly restored the hepatic antioxidants and reduced the pro-inflammatory cytokines in the liver. The antioxidant activity of AB may be attributed to its terpenoid constituents, which was confirmed by LC-Q-TOF-MS/MS analysis. The CCl4-induced rise in expression of MMP-2 and MMP-9 and decrease in TIMP-1 were markedly restored in the AB-treated groups. Further findings revealed a significant reduction in the protein levels of phospho-NF-κB (p65), NLRP3, TGF-ß, pSmad2/3, collagen I, and α-smooth muscle actin (α-SMA) in the AB treatment groups. CONCLUSIONS: The hepatoprotective effect of AB may be attributed to the high content of terpenoid compounds and alleviates liver injury and associated fibrotic changes through modulating MMPs, NF-κB (p65), and the TGF-ß/Smad axis.

Accelerative Wound-Healing Effect of Aqueous Anthocephalus Cadamba Leaf Extract in a Diabetic Rat Model.

Impaired wound healing is a major concern in diabetic patients due to unregulated chronic hyperglycemia which further may lead to ulcer, gangrene, and its complications. The present study unveils the accelerative effect of aqueous Anthocephalus cadamba leaf extract on wound healing in diabetic rats. Diabetes was induced in 30 Sprague Dawley female rats by using streptozotocin (except control group I) at the dose of 60 mg/kg intraperitoneally. Diabetic rats were randomized in 3 groups viz. diabetic control group (II), diabetes + Kadam plant leaf extract group (III), and diabetes + 5% povidone-iodine solution group (IV). Surgically sterile wound of 1.77 cm2 was created on the dorsal area of anaesthetized rats. The experimental parameters were assessed by hematobiochemical, histopathological, and western blot techniques. The A cadamba extract treatment group (III) (D + KPLE) showed a significant increase in the percentage of wound closure (82%) at day 21 as compared to the diabetic control group (42%), nondiabetic control group (I) (49%), and povidone-iodine treatment group (75%) group (IV). The findings of the present study suggest that the (D + KPLE) group (III) exhibited marked epithelial regeneration, neovascularization, collagen deposition, and fibroblast proliferation along with higher expression of vascular endothelial growth factor as compared to the diabetic control group (II), which was confirmed by histopathological examination and western blot analysis. The present study suggests that the topical application of aqueous A cadamba leaf extract exhibits accelerative wound-healing properties in diabetic rats.

A machine learning-based approach to determine infection status in recipients of BBV152 (Covaxin) whole-virion inactivated SARS-CoV-2 vaccine for serological surveys.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

Assessment of antidiabetic effect of 4-HIL in type 2 diabetic and healthy Sprague Dawley rats.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia and insulin resistance. 4-hydroxyisoleucine (4-HIL) is a non-proteinogenic amino acid isolated from the fenugreek seeds and has enormous pharmacological activities. The present study was undertaken to investigate the antihyperglycemic effect of 4-HIL in streptozotocin (STZ)-induced diabetic rats. Moreover, its toxicity was evaluated in vitro and in vivo employing human embryonic kidney cells (HEK-293) and healthy rats, respectively. In experiment 1, STZ-induced diabetic male rats were subjected to an oral treatment of 4-HIL (100 mg/kg), while experiment 2 deals with the effects of 4-HIL on healthy male and female rats following oral administration. The treatment (experiment 1) declined the elevated blood glucose level, feed intake, and increased body weight(s). Additionally, blood glucose impairment was improved as observed by OGTT and IPGT tests. Pancreatic histopathology revealed mild changes in the 4-HIL group. Moreover, experiment 2 showed increased body weight, normal blood glucose levels (male-106.06 ± 7.49 mg/dl and female-100.06 ± 14.69 mg/dL), hematological parameters, and histopathological profiles in the treatment group. 4-HIL did not affect the viability of HEK-293 cells, and no signs of toxicity were observed in healthy rats. Therefore, the study concludes that 4-HIL has potential antihyperglycemic activity without any toxic effects.

In Vitro: Cytotoxicity, Apoptosis and Ameliorative Potential of Lawsonia inermis Extract in Human Lung, Colon and Liver Cancer Cell Line.

Cancer has emerged as a major public health issue in developed as well as in developing countries. Plant-derived molecules are widely being used in the treatment of cancer due to their minimum side effects. Lawsonia inermis (Henna) is one of the medicinal plants containing many therapeutic properties. In the present study, bioactive components of L. inermis extract were analyzed by LCMS/MS method and validated. Lawsone (3.5%) is primarily responsible for cytotoxic and anti-cancerous activities. These properties were studied on human lung carcinoma (A549), colorectal cancer (DLD1) and Hepatocellular carcinoma (HepG2) cancer cell lines. The activities were assessed by MTT assay, evaluation of apoptosis by measuring the production of Reactive Oxygen Species (ROS) and mitochondrial membrane potential of the cancer cell lines. Moreover, apoptosis in the respective cancer cell lines was also determined by chromatin condensation and DNA fragmentation using Hoechst 33528 and propidium iodide (PI) staining. The preliminary in vitro result of MTT showed that the henna extract induces cytotoxic properties against A549, DLD1, HepG2 with IC50values 490, 480 and 610 µg/ml respectively (more than 40% growth inhibition). In addition, the extract induced a concentration-dependent rise in ROS production which was 84, 102, and 110% in HepG2, DLD1 AND A549 respectively at 300 µg/ml, whereas at 400 µg/ml concentration it was 86, 102, and 106% in respective cell lines while decreasing mitochondrial membrane potential was more than 20% in the investigated cell lines. The extract also provoked changes associated with apoptosis and the data indicate that the ROS production leads to a diminution in mitochondrial membrane potential and this correlated with the extract cytotoxicity.