Nimbidiol protects from renal injury by alleviating redox imbalance in diabetic mice.

Introduction: Chronic hyperglycemia-induced oxidative stress plays a crucial role in the development of diabetic nephropathy (DN). Moreover, adverse extracellular matrix (ECM) accumulation elevates renal resistive index leading to progressive worsening of the pathology in DN. Nimbidiol is an alpha-glucosidase inhibitor, isolated from the medicinal plant, 'neem' (Azadirachta indica) and reported as a promising anti-diabetic compound. Previously, a myriad of studies demonstrated an anti-oxidative property of a broad-spectrum neem-extracts in various diseases including diabetes. Our recent study has shown that Nimbidiol protects diabetic mice from fibrotic renal dysfunction in part by mitigating adverse ECM accumulation. However, the precise mechanism remains poorly understood. Methods: The present study aimed to investigate whether Nimbidiol ameliorates renal injury by reducing oxidative stress in type-1 diabetes. To test the hypothesis, wild-type (C57BL/6J) and diabetic Akita (C57BL/6-Ins2Akita/J) mice aged 10-14 weeks were used to treat with saline or Nimbidiol (400 µg kg-1 day-1) for 8 weeks. Results: Diabetic mice showed elevated blood pressure, increased renal resistive index, and decreased renal vasculature compared to wild-type control. In diabetic kidney, reactive oxygen species and the expression levels of 4HNE, p22phox, Nox4, and ROMO1 were increased while GSH: GSSG, and the expression levels of SOD-1, SOD-2, and catalase were decreased. Further, eNOS, ACE2, Sirt1 and IL-10 were found to be downregulated while iNOS and IL-17 were upregulated in diabetic kidney. The changes were accompanied by elevated expression of the renal injury markers viz., lipocalin-2 and KIM-1 in diabetic kidney. Moreover, an upregulation of p-NF-κB and a downregulation of IkBα were observed in diabetic kidney compared to the control. Nimbidiol ameliorated these pathological changes in diabetic mice. Conclusion: Altogether, the data of our study suggest that oxidative stress largely contributes to the diabetic renal injury, and Nimbidiol mitigates redox imbalance and thereby protects kidney in part by inhibiting NF-κB signaling pathway in type-1 diabetes.

Diabetic Nephropathy and Gaseous Modulators.

Diabetic nephropathy (DN) remains the leading cause of vascular morbidity and mortality in diabetes patients. Despite the progress in understanding the diabetic disease process and advanced management of nephropathy, a number of patients still progress to end-stage renal disease (ESRD). The underlying mechanism still needs to be clarified. Gaseous signaling molecules, so-called gasotransmitters, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), have been shown to play an essential role in the development, progression, and ramification of DN depending on their availability and physiological actions. Although the studies on gasotransmitter regulations of DN are still emerging, the evidence revealed an aberrant level of gasotransmitters in patients with diabetes. In studies, different gasotransmitter donors have been implicated in ameliorating diabetic renal dysfunction. In this perspective, we summarized an overview of the recent advances in the physiological relevance of the gaseous molecules and their multifaceted interaction with other potential factors, such as extracellular matrix (ECM), in the severity modulation of DN. Moreover, the perspective of the present review highlights the possible therapeutic interventions of gasotransmitters in ameliorating this dreaded disease.

Glucosidase inhibitor, Nimbidiol ameliorates renal fibrosis and dysfunction in type-1 diabetes.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) leading to renal fibrosis, progressive deterioration of renal function, and eventually to end stage renal disease. Matrix metalloproteinases (MMPs) are known to regulate synthesis and degradation of the ECM. Earlier, we demonstrated that imbalanced MMPs promote adverse ECM remodeling leading to renal fibrosis in type-1 diabetes. Moreover, elevated macrophage infiltration, pro-inflammatory cytokines and epithelial‒mesenchymal transition (EMT) are known to contribute to the renal fibrosis. Various bioactive compounds derived from the medicinal plant, Azadirachta indica (neem) are shown to regulate inflammation and ECM proteins in different diseases. Nimbidiol is a neem-derived diterpenoid that is considered as a potential anti-diabetic compound due to its glucosidase inhibitory properties. We investigated whether Nimbidiol mitigates adverse ECM accumulation and renal fibrosis to improve kidney function in type-1 diabetes and the underlying mechanism. Wild-type (C57BL/6J) and type-1 diabetic (C57BL/6-Ins2Akita/J) mice were treated either with saline or with Nimbidiol (0.40 mg kg-1 d-1) for eight weeks. Diabetic kidney showed increased accumulation of M1 macrophages, elevated pro-inflammatory cytokines and EMT. In addition, upregulated MMP-9 and MMP-13, excessive collagen deposition in the glomerular and tubulointerstitial regions, and degradation of vascular elastin resulted to renal fibrosis in the Akita mice. These pathological changes in the diabetic mice were associated with functional impairments that include elevated resistive index and reduced blood flow in the renal cortex, and decreased glomerular filtration rate. Furthermore, TGF-ß1, p-Smad2/3, p-P38, p-ERK1/2 and p-JNK were upregulated in diabetic kidney compared to WT mice. Treatment with Nimbidiol reversed the changes to alleviate inflammation, ECM accumulation and fibrosis and thus, improved renal function in Akita mice. Together, our results suggest that Nimbidiol attenuates inflammation and ECM accumulation and thereby, protects kidney from fibrosis and dysfunction possibly by inhibiting TGF-ß/Smad and MAPK signaling pathways in type-1 diabetes.

GYY4137 Regulates Extracellular Matrix Turnover in the Diabetic Kidney by Modulating Retinoid X Receptor Signaling.

Diabetic kidney is associated with an accumulation of extracellular matrix (ECM) leading to renal fibrosis. Dysregulation of retinoic acid metabolism involving retinoic acid receptors (RARs) and retinoid X receptors (RXRs) has been shown to play a crucial role in diabetic nephropathy (DN). Furthermore, RARs and peroxisome proliferator-activated receptor γ (PPARγ) are known to control the RXR-mediated transcriptional regulation of several target genes involved in DN. Recently, RAR and RXR have been shown to upregulate plasminogen activator inhibitor-1 (PAI-1), a major player involved in ECM accumulation and renal fibrosis during DN. Interestingly, hydrogen sulfide (H2S) has been shown to ameliorate adverse renal remodeling in DN. We investigated the role of RXR signaling in the ECM turnover in diabetic kidney, and whether H2S can mitigate ECM accumulation by modulating PPAR/RAR-mediated RXR signaling. We used wild-type (C57BL/6J), diabetic (C57BL/6-Ins2Akita/J) mice and mouse mesangial cells (MCs) as experimental models. GYY4137 was used as a H2S donor. Results showed that in diabetic kidney, the expression of PPARγ was decreased, whereas upregulations of RXRα, RXRß, and RARγ1 expression were observed. The changes were associated with elevated PAI-1, MMP-9 and MMP-13. In addition, the expressions of collagen IV, fibronectin and laminin were increased, whereas elastin expression was decreased in the diabetic kidney. Excessive collagen deposition was observed predominantly in the peri-glomerular and glomerular regions of the diabetic kidney. Immunohistochemical localization revealed elevated expression of fibronectin and laminin in the glomeruli of the diabetic kidney. GYY4137 reversed the pathological changes. Similar results were observed in in vitro experiments. In conclusion, our data suggest that RXR signaling plays a significant role in ECM turnover, and GYY4137 modulates PPAR/RAR-mediated RXR signaling to ameliorate PAI-1-dependent adverse ECM turnover in DN.

Crucial role of glucosylceramide synthase in the regulation of stem cell-like cancer cells in B16F10 murine melanoma.

Cancer stem cells render a complex cascade of events that facilitates highly invasive melanoma malignancy. Interplay between immunocytes and cancer stem cells within tumor microenvironment with the participation of sphingolipid signaling mediators skews the immune evasion strategies toward metastatic neoplasm. In this context, we aimed to explore the functional aspect of glucosylceramide synthase (GCS), a key enzyme of sphingolipid biosynthesis in the maintenance of melanoma stem cell-like cancer cells (CSCs). Our findings demonstrated that tumor hypoxia was responsible for elevated GCS expression in melanoma, which was correlated with substantially increased melanoma CSCs. Moreover, hypoxia-induced TGF-ß from TAMs and Tregs promoted GCS induction in B16F10 murine melanoma CSCs via PKCα signaling and facilitated the expansion of melanoma CSCs. Interestingly, GCS ablation hindered the immunosuppressiveness of TAMs and Tregs. Therefore, our study for the first time demonstrated a novel paracrine pathway of melanoma CSC maintenance and tumorigenicity, exploiting the bidirectional signaling with immunocytes. Furthermore, our study showed that the combinatorial immunotherapy involving immunomodulators like Mw and DTA-1 repressed CSC pool affecting GCS functions in advanced-stage B16F10 murine melanoma tumor. Moreover, GCS inhibition sensitized conventional chemotherapeutic drug-resistant melanoma CSCs to the genotoxic drugs paving the way toward selective melanoma treatment. Better therapeutic efficacy with inhibition of GCS and CSC depletion suggests a crucial role of GCS in melanoma treatment, therefore, implying its application concerning clinical challenges of chemotherapy resistance leading to prolonged survival.

Methylation-dependent antioxidant-redox imbalance regulates hypertensive kidney injury in aging.

The prevalence of hypertension increases with age, and oxidative stress is a major contributing factor to the pathogenesis of hypertension-induced kidney damage in aging. The nicotinamide adenine dinucleotide phosphate (NADPH) family is one of the major sources of reactive oxygen species (ROS) generation, and several NADPH oxidase isoforms are highly expressed in the kidney. Although epigenetic protein modification plays a role in organ injury, the methylation of the oxidant-antioxidant defense system and their role in hypertension-induced kidney damage in aging remains underexplored. The present study investigated the role of NADPH oxidase 4, superoxide dismutases (SODs), catalase, and NOS in Ang-II induced kidney damage in aging. Wild type (WT, C57BL/6J) mice aged 12-14 and 75-78 weeks were used and treated with or without Ang-II (1000 ng/kg/min) for 4 weeks with control mice receiving saline. Aged mice with or without Ang-II exhibited higher mean BP, lower renal blood flow, and decreased renal vascular density compared to young mice. While superoxide, 4-HNE, p22phox, Nox4, iNOS were increased in the aged kidney, the expression of eNOS, MnSOD, CuSOD, catalase, Sirt1, and -3 as well as the ratio of GSH/GSSG, and activities of SODs and catalase were decreased compared to young control mice. The changes further deteriorated with Ang-II treatment. In Ang-II treated aged mice, the expressions of DNMTs were increased and associated with increased methylation of SODs, Sirt1, and Nox4. We conclude that hypermethylation of antioxidant enzymes in the aged kidney during hypertension worsens redox imbalance leading to kidney damage.

In vivo experiments demonstrate the potent antileishmanial efficacy of repurposed suramin in visceral leishmaniasis.

BACKGROUND: Treatment failure and resistance to the commonly used drugs remains a major obstacle for successful chemotherapy against visceral leishmaniasis (VL). Since the development of novel therapeutics involves exorbitant costs, the effectiveness of the currently available antitrypanosomatid drug suramin has been investigated as an antileishmanial, specifically for VL,in vitro and in animal model experiments. METHODOLOGY/PRINCIPAL: Leishmania donovani promastigotes were treated with suramin and studies were performed to determine the extent and mode of cell mortality, cell cycle arrest and other in vitro parameters. In addition, L. donovani infected BALB/c mice were administered suramin and a host of immunological parameters determined to estimate the antileishmanial potency of the drug. Finally, isothermal titration calorimetry (ITC) and enzymatic assays were used to probe the interaction of the drug with one of its putative targets namely parasitic phosphoglycerate kinase (LmPGK). FINDINGS: The in vitro studies revealed the potential efficacy of suramin against the Leishmania parasite. This observation was further substantiated in the in vivo murine model, which demonstrated that upon suramin administration, the Leishmania infected BALB/c mice were able to reduce the parasitic burden and also generate the host protective immunological responses. ITC and enzyme assays confirmed the binding and consequent inhibition of LmPGK due to the drug. CONCLUSIONS/SIGNIFICANCE: All experiments affirmed the efficacy of suramin against L. donovani infection, which could possibly lead to its inclusion in the repertoire of drugs against VL.

Glycyrrhizic acid facilitates anti-tumor immunity by attenuating Tregs and MDSCs: An immunotherapeutic approach.

Melanoma is one of the most aggressive malignancies and its treatment remains challenging due to its highly metastatic property and availability of limited effective drugs. In addition, immunosuppresive tumor microenvironment (TME) has been identified as major barrier to evoke anti-tumor response in melanoma. Recent studies revealed that immunosuppressive TME is directly correlated with heightened activations of T regulatory cells (Tregs) and Myeloid derived suppressor cells (MDSCs) functions. In this study, we investigated the anti-cancer effect of a triterpenoid, glycyrrhizic acid (GA) on melanoma. Our study revealed that GA not only exhibited anti-proliferative effects on melanoma cells it significantly restricted progression of melanoma tumor. However, the therapeutic efficacy of GA in impressive regression of tumor was found to be directly correlated with induction of apoptosis and modulation of cytokines from Th2 to Th1 type. To unravel the mechanism of anti-melanoma effect of GA, it has been delineated that GA inhibits pSTAT3 to evade anti-tumor suppressive function of Tregs and MDSCs. Downregulation of FOXP3, GITR and CTLA4 in tumor-infiltrating Tregs and inhibition of Cox2, PGE2 and Arginase 1 in intra-tumoral MDSC were evidenced as some of the key events during therapeutic intervention of GA in melanoma management. Moreover, GA effectively restricted advanced stage solid tumor while used in combination with Mycobacterium indicus pranii, a known immunomodulator, which alone is reported to be ineffective to restrict advanced stage solid tumor. Thus, our findings may open up a novel insight of GA as a promising agent in cancer immunotherapy or adjuvant therapy in future.

Cancer stem cells and ceramide signaling: the cutting edges of immunotherapy.

The multipotent, self renewing "cancer stem cells" (CSCs), a small population within tumor microenvironment facilitates transformed cells to grow and propagate within the body. The CSCs are discovered as resistant to the chemotherapeutic drug with distinct immunological characteristics. In recent years, immunologically targeting CSCs have emerged as an integral part of effective and successful cancer therapy. CSCs notably exhibit dysregulation in conventional sub-cellular sphingolipid metabolism. Recently, ceramide decaying enzymes have been shown to activate alternative ceramide signaling pathways leading to reduction in efficacy of the chemotherapeutic drugs. Therefore, a control over ceramide mediated modulations of CSCs offers an attractive dimension of effective cancer treatment strategy in future. In this review, we focused on the recent findings on broad spectrum of ceramide mediated signaling in CSCs within the tumor niche and their role in potential cancer immunotherapy.

Immunomodulator mediated changes in plasma membrane calcium ATPase in controlling visceral leishmaniasis.

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.

PKCζ mediated anti-proliferative effect of C2 ceramide on neutralization of the tumor microenvironment and melanoma regression.

Immunotherapy, which has advantages over chemotherapy due to lesser toxicity and higher specificity, is on the rise to treat cancer. Recently, pro-apoptotic glycolipid, ceramide has emerged as a key regulator in cancer immunotherapy. The present study elucidated the potential anti-melanoma efficacy of cell-permeable, exogenous C2 ceramide on cell death and amelioration of tumor microenvironment (TME). We, for the first time, demonstrated that C2 ceramide triggered apoptosis of melanoma cells by augmenting PKCζ along with pro-inflammatory cytokines and signaling factors. C2 ceramide showed a PKCζ-mediated tumor-suppressive role in melanoma without exhibiting hepatotoxicity and nephrotoxicity. Moreover, PKCζ was revealed as one of the key regulators of Akt and ceramide during C2 ceramide-mediated apoptosis. C2 ceramide was effective in repolarization of M2 macrophage phenotype and reduction of angiogenic factors such as VEGF, VEGFR1, VEGFR2, HIF1α. Interestingly, PKCζ knockdown attenuated C2 ceramide-mediated inhibition of melanoma progression. Restoration of the Th1 type TME by C2 ceramide enhanced cytotoxic T cell-mediated killing of melanoma cells. Altogether, the study unraveled that C2 ceramide-induced PKCζ was associated with favorable immune cell functioning in TME leading to melanoma regression. Thus, our findings explored a novel mechanistic insight into C2 ceramide as a promising immunotherapeutic agent in melanoma treatment.

TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner.

Ceramide is one of the important cellular components involved in cancer regulation and exerts its pleiotropic role in the protective immune response without exhibiting any adverse effects during malignant neoplasm. Although, the PKCδ-ceramide axis in cancer cells has been an effective target in reduction of cancer, involvement of PKCδ in inducing nephrotoxicity have become a major questionnaire. In the present study, we have elucidated the mechanism by which cisplatin exploits the ceramide to render cancer cell apoptosis leading to the abrogation of malignancy in a PKCδ independent pathway with lesser toxicity. Our study revealed that cisplatin treatment in PKCδ silenced melanoma cells induces ceramide mediated apoptosis. Moreover, cisplatin induced upregulation of the transcription factor IRF1 leading to the induction of the transcriptional activity of the TNFα promoter was evident from the pharmacological inhibition and RNA interference studies. Increased cellular expression of TNFα resulted in an elevated ceramide generation by stimulating acid-sphingomyelinase and cPLA2. Furthermore, reciprocity in the regulation of sphingosine kinase 1 (Sphk1) and sphingosine kinase 2 (Sphk2) during PKCδ independent ceramide generation was also observed during cisplatin treatment. PKCδ inhibited murine melanoma model showed reduction in nephrotoxicity along with tumor regression by ceramide generation. Altogether, the current study emphasized the unexplored signaling cascade of ceramide generation by cisplatin during PKCδ silenced condition, which is associated with increased TNFα generation. Our findings enlightened the detailed mechanistic insight of ceramide mediated signaling by chemotherapeutic drugs in cancer therapy exploring a new range of targets for cancer treatment strategies.

Estrogen-regulated expression of cyp19a1a and cyp19a1b genes in swim-up fry of Labeo rohita.

P450 aromatase is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. The expression of cyp19a1 genes in brain and gonad of Indian major carp, Labeo rohita swim-up fry was measured by quantitative real-time polymerase chain-reaction. Results demonstrated that cyp19a1b and cyp19a1a predominate in brain and gonad respectively. Treatment of fry with an aromatase inhibitor fadrozole for 6days attenuated brain cyp19a1b expression, but not cyp19a1a of gonad. Fadrozole also attenuated brain aromatase activity. Treatment with 17ß-estradiol (E2) for 6days resulted in up-regulation of brain cyp19a1b transcripts in a dose- and time-dependent manner, but not cyp19a1a. Whole-body concentration of vitellogenin also increased in response to E2. Altogether, these results indicate L. rohita swim-up fry can be used to detect environmental estrogens either using vitellogenin induction or cyp19a1b gene expression.

Biological properties of Indian walking catfish (Clarias batrachus) (L.) gonadotropins in female reproduction.

The biological activities of catfish LH-like (semi-purified: s200a and purified Qa) and FSH-like (semi-purified: s200b and purified: Qb) were compared in intact and hypophysectomized female catfish, Clarias batrachus, during preparatory and the pre-spawning periods on vitellogenesis and ovarian maintenance, as well as in vitro final maturation of oocytes, germinal vesicle breakdown (GVBD). During preparatory period, in intact catfish, semi-purified FSH-like induced complete vitellogenesis through the production of estradiol-17ß (E2) and vitellogenin (Vg) accompanied by the formation of SIII yolky oocytes. On the other hand, semi-purified LH-like had induced the formation of only SII (characterized by the appearance of cortical alveoli in cytoplasm) oocytes, which indicates the initiation of vitellogenesis. In hypophysectomized female catfish, purified LH-like but not FSH-like induced the formation of SII oocytes in the ovaries. Treatment with semi-purified LH- and FSH-like at the dose level of 5 µg/fish/day for 7 days significantly maintained the yolky oocytes in gravid catfish after hypophysectomy with a significant reduction in plasma Vg, but not E2 levels, indicating some unknown GtH-induced factor doing the job. In in vitro oocytes culture, both LH- and FSH-like induced GVBD, but the response was significantly more with LH-like than FSH-like. All these findings revealed that both LH-like and FSH-like have overlapping physiological functions, but their responses differ depending on the physiological status of the catfish.