Pralhad Sadashiv Patki

Quercetin ameliorate insulin resistance and up-regulates cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells

Hepatic lipid accumulation and oxidative stress contribute to non-alcoholic fatty liver disease (NAFLD). Thus, we hypothesized that the hypolipidemic and antioxidant activity of quercetin would attenuate events leading to NAFLD. Addition of 2.0mM oleic acid (OA) into the culture media induced fatty liver condition in HepG2 cells by 24h. It was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. The inflammatory cytokines TNF-α and IL-8 levels were significantly increased with decreased antioxidant molecules. OA induced insulin resistance which was evident by inhibition of glucose uptake and cell proliferation. Quercetin (10 μM) increased cell proliferation by 3.05 folds with decreased TAG content (45%) and was effective in increasing insulin mediated glucose uptake by 2.65 folds. The intracellular glutathione content was increased by 2.0 folds without substantial increase in GSSG content. Quercetin (10 μM) decreased TNF-α and IL-8 by 59.74% and 41.11% respectively and inhibited generation of lipid peroxides by 50.5%. In addition, RT-PCR results confirmed quercetin (10 μM) inhibited TNF-alpha gene expression. Further, superoxide dismutase, catalase and glutathione peroxidase activities were increased by 1.68, 2.19 and 1.71 folds respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by quercetin. Hence, quercetin effectively reversed NAFLD symptoms by decreased triacyl glycerol accumulation, insulin resistance, inflammatory cytokine secretion and increased cellular antioxidants in OA induced hepatic steatosis in HepG2 cells.

Liv.52 attenuate copper induced toxicity by inhibiting glutathione depletion and increased antioxidant enzyme activity in HepG2 cells.

Altered copper metabolism plays a pivotal role in the onset of several hepatic disorders and glutathione (GSH) plays an important role in its homeostasis. Hepatic diseases are often implicated with decreased content of intracellular GSH. GSH depleted cells are prone to increased oxidative damage eventually leading to its death. Liv.52 is used to treat hepatic ailments since long time. Hence, in the present study the potential cytoprotective effect of Liv.52 against toxicity induced by copper (Cu2+) was evaluated in HepG2 cells. Cu2+ at 750 microM induced cytotoxicity to HepG2 cells as determined by MTT assay. The toxicity was brought about by increased lipid peroxidation, DNA fragmentation and decreased GSH content. But, upon treatment with Liv.52 cell death induced by Cu2+ was significantly abrogated by inhibition of lipid peroxidation by 58% and DNA fragmentation by 37%. Liv.52 increased the GSH content by 74%. Activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase were increased by 46%, 22% and 81% respectively in Liv.52 treated cells. Thus, it is apparent from these results that Liv.52 abrogates Cu2+ induced cytotoxicity in HepG2 cells by inhibiting lipid peroxidation and increased GSH content and antioxidant enzyme activity.

Alcohol depletes coenzyme-Q10 associated with increased TNF-alpha secretion to induce cytotoxicity in HepG2 cells

Alcohol consumption has been implicated to cause severe hepatic steatosis which is mediated by alcohol dehydrogenase (ADH) activity and CYP(450) 2E1 expression. In this context, the effect of ethanol was studied for its influence on lipogenesis in HepG2 cell which is deficient of ADH and does not express CYP(450) 2E1. The results showed that ethanol at 100mM concentration caused 40% cytotoxicity at 72h as determined by MTT assay. The incorporation of labeled [2-(14)C] acetate into triacylglycerol and phospholipid was increased by 40% and 26% respectively upon 24h incubation, whereas incorporation of labeled [2-(14)C] acetate into cholesterol was not significantly increased. Further, ethanol inhibited HMG-CoA reductase which is a rate-limiting enzyme in the cholesterol biosynthesis. It was observed that, HMG-CoA reductase inhibition was brought about by ethanol as a consequence of decreased cell viability, since incubation of HepG2 cells with mevalonate could not increase the cholesterol content and increase the cell viability. Addition of ethanol significantly increased TNF-alpha secretion and depleted mitochondrial coenzyme-Q(10) which is detrimental for cell viability. But vitamin E (10mM) could partially restore coenzyme-Q(10) and glutathione content with decreased TNF-alpha secretion in ethanol treated cells. Further, lipid peroxidation, glutathione peroxidase and superoxide dismutase enzyme activities remained unaffected. Ethanol decreased glutathione content while, GSH/GSSG ratio was significantly higher compared to other groups showing cellular pro-oxidant and antioxidant balance remained intact. Alanine amino transferase activity was increased by 4.85 folds in cells treated with ethanol confirming hepatocyte damage. Hence, it is inferred that ethanol induced cytotoxicity in HepG2 cells due to coenzyme-Q(10) depletion and increased TNF-alpha secretion.

Anti-inflammatory properties of Septilin in lipopolysaccharide activated monocytes and macrophage

In vivo studies have suggested the immunomodulatory properties of Septilin, an herbal preparation. This drug is being used against various types of inflammatory disorders. However, the mechanism of action of Septilin in the modulation of inflammation is not explored using suitable in vitro models. Hence, we decided to study the modulatory role of Septilin in lipopolysaccharide (LPS) mediated signaling in macrophage and monocyte cells. It was observed from the present study that by employing tumor necrosis factor α (TNF-α) bioassay and reverse transcription-polymerase chain reaction (RT-PCR), Septilin inhibited TNF-α production in LPS (1 μg/mL) stimulated RAW 264.7 cells (p < 0.05). 80% inhibition of TNF-α was observed even at 2.5% Septilin. Septilin at all the concentrations tested could also significantly block the LPS mediated nitric oxide (NO) production (p < 0.01) and expression of inducible NO synthase (iNOS) gene. LPS mediated interleukin 6 (IL-6) and IL-8 production was also blocked by Septilin at the concentrations tested. This herbal preparation could also inhibit cycloxygenase 2 (COX-2) activity and suppression of COX-2 and phosphodiesterase 4 B (PDE4B) mRNA expression in a concentration dependent manner. Taken together, these findings from the present in vitro study suggest the anti-inflammatory and immunomodulatory properties of Septilin.

Yashada bhasma (Zinc calx) and Tankana (Borax) inhibit Propionibacterium acne and suppresses acne induced inflammation in vitro

Yashada bhasma (YB) and Tankana (TA) are well characterized minerals used in traditional medicine for the treatment of various skin ailments. Yashada bhasma and TA are a unique preparation of zinc and borax, respectively. The study was conducted to evaluate the in vitro inhibitory effect of YB, TA and its combination (YBTA) on Propionibacterium acne growth and P. acne‐induced inflammation.

Liv.52 up-regulates cellular antioxidants and increase glucose uptake to circumvent oleic acid induced hepatic steatosis in HepG2 cells.

HepG2 cells were rendered steatotic by supplementing 2.0mM oleic acid (OA) in the culture media for 24h. OA induced hepatic steatosis in HepG2 cells was marked by significant accumulation of lipid droplets as determined by Oil-Red-O (ORO) based colorimetric assay, increased triacylglycerol (TAG) and increased lipid peroxidation. It was also marked by increased inflammatory cytokines TNF-α and IL-8 with decreased enzymic and non-enzymic antioxidant molecules and decreased cell proliferation associated with insulin resistance and DNA fragmentation. Addition of Liv.52 hydro-alcoholic extract (LHAE) 50μg/mL to the steatotic cells was effective in increasing the insulin mediated glucose uptake by 3.13 folds and increased cell proliferation by 3.81 folds with decreased TAG content (55%) and cytokines. The intracellular glutathione content was increased by 8.9 folds without substantial increase in GSSG content. LHAE decreased TNF-α and IL-8 by 51% and 6.5% folds respectively, lipid peroxidation by 65% and inhibited DNA fragmentation by 69%. The superoxide dismutase, catalase and glutathione peroxidase activities were increased by 88%, 128% and 64% respectively. Albumin and urea content was increased while the alanine aminotransferase (ALAT) activity was significantly decreased by LHAE. Hence, LHAE effectively attenuate molecular perturbations associated with non-alcoholic fatty liver disease (NAFLD) indications in HepG2 cells.

HD-03/ES: A promising herbal drug for HBV antiviral therapy

INTRODUCTION The present study was designed to study the genotypes associated with different groups of chronic liver disease and to see their response to HD-03/ES (an antiviral herbal molecule) on chronic HBV patients. METHODS A total of 51 patients of chronic liver disease were recruited in the study and were given HD-03/ES, two capsules twice daily for 6 months. Liver function tests were done every month after initiating treatment. Serum was analyzed for HBsAg, HBeAg and HBV DNA and quantitative estimation of HBV at baseline, 4 and 6 months after therapy. The genotype of all the cases was also determined by PCR-RFLP method. RESULTS After 6 months of therapy with HD-03/ES, a significant reduction of ALT values from 71.2 + or - 16.3 to 36.4 + or - 6.8 and a significant HBeAg loss (27.4%) and HBV DNA loss (27.4%) was observed. Adverse effects were mild. Genotype D was found in 39 (76.5%) while genotype A was found in 12 (33.5%) cases, respectively. The mean reduction in viral load was observed from log(10) 7.1 + or - 1.8 copies/ml to log(10) 4.4 + or - 1.1 copies/ml. However, a sharp decline in viral load was observed in patients infected with genotype A (log(10) 6.8 + or - 2.5 to log(10) 4.9 + or - 1.8; P < 0.01) compared to genotype D (log(10) 7.0 + or - 2.6 to log(10) 5.9 + or - 3.5; P = 0.074). CONCLUSION The study had shown that majority of the patients of chronic HBV related liver disease had genotype D. In additional, the molecule HD03/ES had a better therapeutic capability of lowering the HBV viral load in patients with genotype A, which needs to be validated in larger studies.

Ethanol extract of Justicia gendarussa inhibits lipopolysaccharide stimulated nitric oxide and matrix metalloproteinase-9 expression in murine macrophage.

Context: Justicia gendarussa Burm (Acanthaceae) is a plant used to treat inflammatory diseases such as rheumatoid arthritis. However, the mechanism involved in the anti-inflammatory properties of this plant has not been studied well. Objective: The in vitro anti-inflammatory activities of ethanol extract of Justicia gendarussa leaves (J-01) are studied here for the first time. Materials and methods: The ethanol extract, J-01 was prepared from the leaves of Justicia gendarussa. The inhibitory effect of J-01 in nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9) gene expressions were studied in lipopolysaccharide (LPS) stimulated macrophage cell line RAW 264.7. Results: J-01 in a concentration dependent manner (200–50 μg/mL) attenuated NO production from macrophage stimulated with LPS (1 μg/mL). Further, J-01 significantly suppressed iNOS mRNA expression in these cells. J-01 has also downregulated the MMP-9 gene expression in LPS stimulated macrophage. Discussion and conclusion: The modulatory function of J-01 in inhibiting NO, iNOS, and MMP-9 as obtained from the present in vitro studies provide first scientific evidence to support the anti-inflammatory properties of Justicia gendarussa. This plant may have potential use in the management of inflammatory conditions such as arthritis.

Bresol inhibits phosphodiesterase 4 gene expression and modulates the levels of select mediators of inflammation in human monocytic cells

Bresol–a poly-herbal formulation, has been reported to be effective against bronchial asthma and allergic rhinitis in children. In vivo studies have supported the anti-histaminic and anti-anaphylactic action of bresol. However, the mechanism of action of bresol in modulation of inflammation has not been studied at the cellular and molecular level. The present study was aimed to elucidate the mechanism(s) of action of bresol at the cellular and molecular levels, using human monocyte leukemia cells. The effects of bresol on phosphodiesterase 4B (PDE4B) gene expression were analyzed using human monocytic U937 leukemia cells. The ability of bresol to stimulate cAMP formation in these cells, as well as its effects on mediators of inflammation like tumor necrosis factor-α (TNFα), nitric oxide (NO), and cycloxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated U937 cells, were also studied. The results here indicated that bresol exhibited potential anti-inflammatory properties by inhibiting LPS-induced PDE4B gene expression in the cells. Bresol also dose dependently activated cAMP formation, and inhibited TNFα, NO, as well as COX-2 formation in the LPS-stimulated cells. Based upon the results, we concluded that the reported anti-inflammatory activity of bresol might be attributed to its abilities to inhibit PDE4B and thus elevate cAMP levels in human monocytes. The anti-inflammatory effects of bresol might also be a result of the capacity of bresol to modulate the formation of TNFα, NO, and COX-2 in monocytes.

Water Soluble Components of 'Osteocare' Promote Cell Proliferation, Differentiation, and Matrix Mineralization in Human Osteoblast-Like SaOS-2 Cells

Osteocare, a herbal formulation, has been found to be very effective in bone mineralization and support of the microstructure of bone tissue. The water-soluble components of Osteocare (WSCO) induced osteogenic activity in human osteoblast-like SaOS-2 cells. The addition of WSCO (100 μg/ml) to SaOS-2 cells was effective in increasing the cell proliferation by 41.49% and DNA content by 1.9-fold. WSCO increased matrix mineralization in SaOS-2 cells by increased alkaline phosphatase levels and calcium-rich deposits as observed by Alizarin red staining. WSCO markedly increased mRNA expression for osteopontin (OPN), osteocalcin (OCN), type I collagen (Col I) in SaOS-2 cells, and it down-regulated IL-6 mRNA levels in SaOS-2 cells. The present study showed that WSCO plays an important role in osteoblastic bone formation through enhanced activities of ALP, Col I, bone matrix proteins such as OPN and OCN, down-regulation of cytokines like IL-6, as well as promoting mineralization in SaOS-2 cells.