Vishakha Singh

TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery

The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K(+) depolarizing solution or K(+) channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10(-10)-10(-7)M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.

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.

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-β1), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), interleukin-1 beta (IL-1β) 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-β1, 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-1β 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.

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-β1()), tumor necrosis factor-α (TNF-α) and interlukin-10 (IL-10) mRNA and proteins and the mRNA of interlukin-1 beta (IL-1β) 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-β1, 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-1β 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.

Combined treatment with atorvastatin and imipenem improves survival and vascular functions in mouse model of sepsis

We have recently reported that pre-treatment, but not the post-treatment with atorvastatin showed survival benefit and improved hemodynamic functions in cecal ligation and puncture (CLP) model of sepsis in mice. Here we examined whether combined treatment with atorvastatin and imipenem after onset of sepsis can prolong survival and improve vascular functions. At 6 and 18h after sepsis induction, treatment with atorvastatin plus imipenem, atorvastatin or imipenem alone or placebo was initiated. Ex vivo experiments were done on mouse aorta to examine the vascular reactivity to nor-adrenaline and acetylcholine and mRNA expressions of α1D AR, GRK2 and eNOS. Atorvastatin plus imipenem extended the survival time to 56.00±4.62h from 20.00±1.66h observed in CLP mice. The survival time with atorvastatin or imipenem alone was 20.50±1.89h and 27.00±4.09h, respectively. The combined treatment reversed the hyporeactivity to nor-adrenaline through preservation of α1D AR mRNA/protein expression and reversal of α1D AR desensitization mediated by GRK2/Gβγ pathway. The treatment also restored endothelium-dependent relaxation to ACh through restoration of aortic eNOS mRNA expression and NO availability. In conclusion, combined treatment with atorvastatin and imipenem exhibited survival benefit and improved vascular functions in septic mice.

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.

NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2

BACKGROUND The aim of the present study was to observe the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery and explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery. METHODS Tension experiments were conducted on the pulmonary artery from male Wistar rats. RESULTS TRPV4 channel agonist GSK1016790A (GSK) caused concentration-dependent relaxation (Emax 86.9±4.6%; pD2 8.7±0.24) of the endothelium-intact rat pulmonary artery. Combined presence of apamin and TRAM-34 significantly attenuated the relaxation (Emax 61.1±6.0%) to GSK. l-NAME (100μM) significantly attenuated (8.2±2.9%) the relaxation response to GSK that was resistant to apamin plus TRAM-34. However, presence of ICI192605 or furegrelate alongwith l-NAME revealed the GSK-mediated EDHF-response (Emax of 28.5±5.2%; Emax 24.5±4.3%) in this vessel, respectively. Further, these two TxA2 modulators (ICI/furegrelate) alongwith l-NAME had no effect on SNP-induced endothelium-independent relaxation in comparison to l-NAME alone. This EDHF-mediated relaxation was sensitive to inhibition by K(+) channel blockers apamin and TRAM-34 or 60mMK(+) depolarizing solution. Further, combined presence of apamin and TRAM-34 in U46619 pre-contracted pulmonary arterial rings significantly reduced the maximal relaxation (Emax 71.6±6.9%) elicited by GSK, but had no effect on the pD2 (8.1±0.03) of the TRPV4 channel agonist in comparison to controls (Emax, 92.4±4.3% and pD2, 8.3±0.06). CONCLUSION The present study suggests that NO and EDHF are released concomitantly and NO synthase inhibition attenuates GSK-induced EDHF response through thromboxane pathway in the rat pulmonary artery.

Atorvastatin along with imipenem attenuates acute lung injury in sepsis through decrease in inflammatory mediators and bacterial load

Lung is one of the vital organs which is affected during the sequential development of multi-organ dysfunction in sepsis. The purpose of the present study was to examine whether combined treatment with atorvastatin and imipenem could attenuate sepsis-induced lung injury in mice. Sepsis was induced by caecal ligation and puncture. Lung injury was assessed by the presence of lung edema, increased vascular permeability, increased inflammatory cell infiltration and cytokine levels in broncho-alveolar lavage fluid (BALF). Treatment with atorvastatin along with imipenem reduced the lung bacterial load and pro-inflammatory cytokines (IL-1β and TNFα) level in BALF. The markers of pulmonary edema such as microvascular leakage and wet-dry weight ratio were also attenuated. This was further confirmed by the reduced activity of MPO and ICAM-1 mRNA expression, indicating the lesser infiltration and adhesion of inflammatory cells to the lungs. Again, expression of mRNA and protein level of iNOS in lungs was also reduced in the combined treatment group. Based on the above findings it can be concluded that, combined treatment with atorvastatin and imipenem dampened the inflammatory response and reduced the bacterial load, thus seems to have promising therapeutic potential in sepsis-induced lung injury in mice.