Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice.

Sepsis commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity and mortality. Septic ALI is characterized by excessive production of proinflammatory mediators. It remained refractory to present therapies and new therapies need to be developed to improve further clinical outcomes. Betulinic acid (BA), a pentacyclic lupane group triterpenoid has been shown to have anti-inflammatory activities in many studies. However, its therapeutic efficacy in polymicrobial septic ALI is yet unknown. Therefore, we investigated the effects of BA on septic ALI using cecal ligation and puncture (CLP) model in mice. Vehicle or BA (3, 10, and 30mg/kg) was administered intraperitoneally, 3 times (0, 24 and 48h) before CLP and CLP was done on 49(th)h of the study. Survival rate was observed till 120h post CLP. Lung tissues were collected for analysis by sacrificing mice 18h post CLP. BA at 10 and 30mg/kg dose significantly reduced sepsis-induced mortality and lung injury as implied by attenuated lung histopathological changes, decreased protein and neutrophils infiltration. BA also decreased lung NF-κB expression, cytokine, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and matrix metalloproteinase-9 levels. These evidences suggest that, the protective effects of BA on lungs are associated with defending action against inflammatory response and BA could be a potential modulatory agent of inflammation in sepsis-induced ALI.

Betulinic acid negates oxidative lung injury in surgical sepsis model.

BACKGROUND: Sepsis commonly progresses to acute lung injury and is associated with high morbidity and mortality. Septic acute lung injury is characterized by severe oxidative stress response, remained refractory to present therapies, and new therapies need to be developed to improve further clinical outcomes. We determined the effect of betulinic acid (BA) on oxidative lung injury in mice using cecal ligation and puncture (CLP) model. MATERIALS AND METHODS: Five groups of mice (six in each group) received three pretreatments at 24-h interval before surgery. Surgery was done 1 h after last dosing. Sham and CLP control group mice received vehicle. BA was administered to other three groups of mice at 3, 10, and 30 mg/kg dose. Lung and plasma samples were collected for analysis by sacrificing the mice at 18 h of surgery. RESULTS: Compared with sham, CLP significantly increased total protein, nitrite, malondialdehyde, isoprostane, superoxide, protein carbonyl, oxidative stress index, inducible nitric oxide synthase protein, and histopathologic changes and reduced the superoxide dismutase, catalase activity, and total thiol levels in lungs and plasma, which were restored by BA pretreatment. CONCLUSIONS: BA pretreatment decreased the levels of oxidants, increased the levels of antioxidants in lungs and plasma thereby reducing the oxidative lung injury in CLP mice. Additionally, BA was found to scavenge the superoxide and nitric oxide radical in vitro. Thus, BA is suggested to be effective in treatment of oxidative lung injury in sepsis.

Curcumin-induced angiogenesis hastens wound healing in diabetic rats.

BACKGROUND: Neovasculogenesis, vital for wound healing, gets compromised in diabetics patients, which consequently delayed wound healing. Previous studies have shown curcumin as both a stimulatory and an inhibitory agent in the neovasculogenesis process. So, present study was aimed to investigate the effects of curcumin on wound healing in diabetic rats and to explore the expressions of the various factors involved in neovasculogenesis. MATERIALS AND METHODS: Open excisional diabetic wound was created in sixty rats and divided into three groups viz. i) control, ii) pluronic gel-treated, and iii) curcumin-treated. The pluronic F-127 gel (25%) and curcumin (0.3%) in the pluronic gel were topically applied once daily for 19 d. The wound healing and neovasculogenesis among these groups were evaluated by gross appearance of wounds and microscopically by hematoxylin and eosin staining, immunohistochemistry for CD31, messenger RNA expressions of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1, hypoxia-inducible growth factor-1 alpha, stromal cell-derived growth factor-1 alpha, and heme oxygenase-1, and Western blotting studies of VEGF and TGF-ß1 in granulation and/or healing tissue on days 3, 7, 14, and 19. RESULTS: Curcumin application caused markedly fast wound closure with well-formed granulation tissue dominated by fibroblast proliferation, collagen deposition, and complete early regenerated epithelial layer. Immunohistochemistry for CD31 revealed well-formed blood vessels with increased microvessel density on days 3, 7, and 14 in the curcumin-treated group. Expressions of VEGF and TGF-ß1 on days 3, 7, and 14, hypoxia-inducible growth factor-1 alpha, stromal cell-derived growth factor-1 alpha, and heme oxygenase-1 on days 3 and 7 were increased in curcumin-treated diabetic rats, as compared with other groups. CONCLUSIONS: Curcumin enhanced the neovasculogenesis and accelerated the wound healing in diabetic rats by increased expressions of various factors.

Hemin attenuated oxidative stress and inflammation to improve wound healing in diabetic rats.

Oxidative stress and persistent inflammation play crucial role in the progression of diabetic wound complications. Hemeoxgenase-1 (HO-1) by degrading hemin has been shown to display anti-oxidant and anti-inflammatory effects. Further, hemin is a potent HO-1 inducer. Thus, the current study was aimed to evaluate the effect of topical application of hemin on diabetic wound in rats. Four hundred square millimeter open excision wound were created 2 weeks after induction of diabetes with single intraperitoneal injection of streptozotocin (60 mg/kg), and the diabetic rats were divided into three groups namely diabetic control, hemin, and tin protoporphyrin (SnPPIX). Ointment base, hemin (0.5% in ointment base), and SnPPIX (0.5% in ointment base) were applied topically to wounded area in diabetic control, hemin, and SnPPIX group rats, respectively, twice daily for 19 days. Hemin significantly increased the wound contraction in comparison to control and SnPPIX-treated rats. Time-dependent analysis revealed significant increase in anti-oxidants with concomitant decrease in oxidants in hemin-treated rats as compared to diabetic control rats. Further, mRNA expression decreased for inflammatory cytokine and increased for anti-inflammatory cytokine in hemin group as compared to diabetic control rats. Expression of HO-1 also increased in hemin group as compared to diabetic control rats. However, SnPPIX group results were in disagreement with results of hemin which is clearly reflected in histopathology. Results indicate the ability of hemin to accelerate wound healing in diabetic rats by combating inflammation and oxidative stress probably via HO-1.

Phosphorylation: Implications in Cancer.

Post translational modifications (PTMs) are involved in variety of cellular activities and phosphorylation is one of the most extensively studied PTM, which regulates a number of cellular functions like cell growth, differentiation, apoptosis and cell signaling in healthy condition. However, alterations in phosphorylation pathways result in serious outcomes in the form of diseases, especially cancer. Many signalling pathways including Tyrosine kinase, MAP kinase, Cadherin-catenin complex, Cyclin-dependent kinase etc. are major players of the cell cycle and deregulation in their phosphorylation-dephosphorylation cascade has been shown to be manifested in the form of various types of cancers. Tyrosine kinase family encompasses the greatest number of oncoproteins. MAPK cascade has an importance role in cancer growth and progression. Bcl-2 family proteins serve either proapoptotic or antiapoptotic function. Cadherin-catenin complex regulates cell adhesion properties and cyclins are the key regulators of cell cycle. Altered phosphorylations in any of the above pathways are strongly associated with cancer, at the same time they serve as the potential tergets for drug development against cancer. Drugs targeting tyrosine kinase are potent anticancer drugs. Inhibitors of MEK, PI3K and ERK signalling pathways are undergoing clinical trials. Thus, drugs targeting phosphorylation pathways represent a promising area for cancer therapy.

Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats.

Bilirubin has shown cutaneous wound healing potential in some preliminary studies. Here we hypothesize that bilirubin facilitates wound healing in diabetic rats by modulating important healing factors/candidates and antioxidant parameters in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin. In all diabetic rats wounds were created under pentobarbitone anesthesia. All the rats were divided into two groups, of which one (control) was treated with ointment base and other with bilirubin ointment (0.3%). Wound closer measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 alpha (SDF-1α), transforming growth factor- beta1 (TGF-ß1()), tumor necrosis factor-α (TNF-α) and interlukin-10 (IL-10) mRNA and proteins and the mRNA of interlukin-1 beta (IL-1ß) and matrix metalloprteinase-9 (MMP-9) were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histopathological changes were assessed by H&E staining. The per cent wound closer was significantly higher from day 7 onwards in bilirubin-treated rats. HIF-1α, VEGF, SDF-1α, TGF-ß1, IL-10 mRNA and protein levels were significantly higher on days 3, 7 and 14 in bilirubin-treated rats. The mRNA expression and protein level of TNF-α and the mRNA of IL-1ß and MMP-9 were progressively and markedly reduced in bilirubin-treated rats. The collagen deposition and formation of blood vessels were greater in bilirubin-treated rats. Bilirubin markedly facilitated cutaneous wound healing in diabetic rats by modulating growth factors, cytokines, neovasculogenesis and collagen contents to the wound site. Topical application of bilirubin ointment might be of great use in cutaneous wound healing in diabetic patients.

Molecular and functional characterization of TRPV4 channels in pregnant and nonpregnant mouse uterus.

AIMS: The aim of the present study was to characterize TRPV4 channels in pregnant and nonpregnant mouse uterus and examine their functional role in spontaneous and agonist-induced contractions. MAIN METHODS: We used RT-PCR, Western blot and immunohistochemistry experiments to demonstrate the presence of TRPV4 mRNA and protein, respectively in both pregnant and nonpregnant mouse uterus. Tension experiments were conducted for functional characterization of the TRPV4 channels. KEY FINDINGS: TRPV4 mRNA and protein were detected in both pregnant and nonpregnant mouse uterus with distribution in both endometrium and myometrium. The TRPV4 channel agonist GSK1016790A (GSK) increased myometrial contraction in pregnant (Emax 336.8±21.35%; pD2 7.79±0.29) and nonpregnant (Emax 238±28.13%; pD2 7.61±0.57) animals. HC067047 (1µM), a selective blocker of the TRPV4 channel, antagonized the contractions to GSK in pregnant (Emax 171±18.26%; pD2 6.58±0.37) and nonpregnant (Emax 78.12±9.32%; pD2 7.54±0.9) uteri. Further, HC067047 (1µM) inhibited contractions induced by PGF2α in the pregnant (Emax 183.2±13.94%; pD2 7.01±0.30 versus control Emax 495.7±42.49%; pD2 7.12±0.24) and nonpregnant (Emax 105.3±7.10%; pD2 7.24±0.34 versus control Emax 232.5±12.27%; pD2 7.83±0.29) uteri. SIGNIFICANCE: TRPV4 channels are present in the pregnant and nonpregnant mouse uteri, and their activation by endogenous ligands like prostaglandin increases myometrial contractility. Thus, the TRPV4 channel can be an important target in reducing myometrial contractility in preterm labor.

Deferoxamine modulates cytokines and growth factors to accelerate cutaneous wound healing in diabetic rats.

Deferoxamine has shown cutaneous wound healing potential by increased neovascularization. We hypothesized that topically applied deferoxamine facilitates wound healing in diabetic rats by modulating important cytokines and growth factors that take part in healing processes in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin and wound was created under pentobarbitone anesthesia. The diabetic rats were divided into two groups, of which one (control) was treated with ointment base and other with deferoxamine ointment (0.1%). Wound closure measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1-alpha (SDF-1α), transforming growth factor beta 1 (TGF-ß1), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), interleukin-1 beta (IL-1ß) and interleukin-10 (IL-10) mRNA and proteins were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histological changes were assessed by H&E staining. The per cent wound closure was significantly higher from day 7 onwards in deferoxamine-treated rats. HIF-1α, VEGF, SDF-1α, TGF-ß1, IL-10 mRNA and their protein levels were significantly higher on days 3, 7 and 14 in deferoxamine-treated rats. The mRNA expression and protein levels of TNF-α, MMP-9 and IL-1ß were progressively and markedly reduced in deferoxamine-treated rats. The collagen deposition and formation of blood vessels were greater in deferoxamine-treated rats. It is suggested that topical application of deferoxamine ointment might be useful in cutaneous wound healing in diabetic patients.

Betulinic acid attenuates renal oxidative stress and inflammation in experimental model of murine polymicrobial sepsis.

Sepsis is a common cause of acute kidney injury (AKI) and is associated with substantial morbidity and mortality. Objective of the study was to evaluate the effect of betulinic acid, a triterpenoid in sepsis-induced AKI using cecal ligation puncture (CLP) mouse model. Mice subjected to CLP developed histologic AKI at 18h after CLP. There was an increase in renal proinflammatory response (nuclear factor-kappa B expression, tumor necrosis factor-alpha, interleukin (IL)-6 and IL-10), matrix metalloproteinase-9, plasma creatinine, renal neutrophil gelatinase-associated lipocalin and oxidant stress response (malondialdehyde, inducible nitric oxide synthase, total nitrite and superoxide); decrease in anti-oxidant levels (superoxide dismutase and catalase) at 18h of CLP. However, BA pretreatment at the doses of 10 and 30mg/kg prevented the CLP-induced kidney damage by restoring the aforementioned inflammatory mediators, oxidant and anti-oxidant imbalance. These evidences suggest that, the protective effects of BA on kidney are associated with defending action against inflammatory and oxidative stress response in CLP mice and BA could be potential therapeutic agent in sepsis-induced AKI.

Antioxidant potential of bilirubin-accelerated wound healing in streptozotocin-induced diabetic rats.

Oxidative injury is markedly responsible for wound complications in diabetes mellitus. The biological actions of bilirubin may be relevant to prevent oxidant-mediated cell death, as bilirubin application at a low concentration scavenges reactive oxygen species. Hence, we hypothesized that topical bilirubin application might improve wound healing in diabetic rats. Diabetes was induced in adult male Wistar rats, which were divided into two groups, i.e., diabetic control and diabetic treated. Non-diabetic healthy rats were also taken as healthy control group. Wound area was measured on days 3, 7, 14, and 19 post-wounding. The levels of malondialdehyde (MDA) and reduced glutathione (GSH) and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were estimated in the granulation tissue. There was a significant increase in percent wound closure in healthy control and diabetic treated rats on days 7, 14, and 19, as compared to diabetic control rats on days 7, 14, and 19. There was significant decrease in MDA levels on days 7, 14, and 19 in diabetic treated rats, as compared to diabetic control rats. Levels of GSH were significantly increased on days 3, 7, 14, and 19 in diabetic treated rats, as compared to diabetic control rats. GPx, SOD, and CAT activities were significantly higher on days 3, 7, and 14 in diabetic treated rats, as compared to diabetic control rats. The findings indicate that bilirubin is effective in reducing the oxidant status in wounds of diabetic rats which might have accelerated wound healing in these rats.

Effect of chronic fenvalerate intoxication on tissue concentration of copper in goats and further exploration of its mechanism.

This experiment was designed to assess the effect of chronic fenvalerate toxicity on tissue Cu concentration in goats and to explore the pathways responsible for it. A significant decrease in tissue Cu concentration of kidney, heart, and brain while an increase in the liver were recorded in fenvalerate intoxicated goats at 15 mg/kg b.w. orally daily for 270 days. Concentration of total Cu, protein-free Cu, and protein-bound Cu in the wet intestine of fenvalerate-treated goats revealed a significant decrease in Cu concentration of the intestine due to the decrease in trichloroacetic acid (TCA)-insoluble Cu, while TCA-soluble Cu remained almost unaffected. Rabbit duodenal loop technique was used to assess the relative absorption of nonisotopic copper in a living animal. This technique enabled to compare Cu absorption from the lumen of three closely associated loops, each receiving 100 µg of copper along with different doses (0, 15, and 30 µg) of fenvalerate. A significant dose-dependent decrease in Cu absorption from the lumen due to fenvalerate treatment was recorded. A decrease in total copper (TCA-insoluble fraction) suggested an interference in active transport of copper due to the inhibition of absorption of protein-bound copper. It was concluded that fenvalerate interfered in copper absorption mostly by inhibiting its active or mediated transport.

Antidiabetic activity and phytochemical screening of crude extract of Stevia rebaudiana in alloxan-induced diabetic rats.

BACKGROUND: Stevia rebaudiana regulates blood sugar, prevents hypertension and tooth decay. Other studies have shown that it has antibacterial as well as antiviral property. METHODS: Preliminary phytochemical screening of aqueous, ether and methanolic extracts of S. rebaudiana was done. Acute and sub-acute toxicity were conducted on twenty four Albino rats, divided into one control (Group I) and three treatment groups viz. aqueous extract (Group II), ether extract (Group III) and methanolic extract (Group IV). For the study of antidiabetic effect of S. rebaudiana rats were divided into seven groups (n=6). Diabetes was induced by a single dose of 5% alloxan monohydrate (125 mg/kg, i.p.) after 24 hour fasting.Blood samples were analysed on day 0, 1, 5, 7, 14 and 28. RESULTS: Phytochemical tests showed presence of different kinds of phyto-constituents in aqueous, ether and methanol extract of Stevia rebaudiana leaves. Daily single dose (2.0 g/kg) administration of aqueous extract (A.E.) , ether extract (E.E.) and methanol extract (M.E.) for 28 days of S. rebaudiana could not show any significant change in ALT and AST levels in rats. Blood sugar level was found to be decreased on day 28 in groups of rats treated with A.E., E.E. and M.E. of S. rebaudiana. CONCLUSION: The extracts of Stevioside rebaudiana could decrease the blood glucose level in diabetic rats in time dependent manner.