Pharmacological blockade of HDAC3 accelerates diabetic wound healing by regulating macrophage activation.

AIMS: Here, we report the effect of histone deacetylase 3 (HDAC3) inhibition associated with macrophage activation, IL-1ß expression, angiogenesis and wound healing in diabetic mice. MAIN METHODS: To determine the expression of HDAC3 in diabetic mice wounds, hyperglycemia was induced in C57BL/6 mice with streptozotocin followed by induction of 6 mm wounds. To understand the effect of HDAC3 selective inhibitor, BG45, wound tissues were isolated for analysing M1/M2 markers expression, immune cells infiltration, angiogenesis and healing factors expression. CD11b+F4/80+ cells were sorted from the wound tissues and analysed for the expression of M1/M2 markers using RT-qPCR and flow cytometer. In cell based assays, HDAC3 expression was measured in macrophages stimulated with high glucose (HG) plus LPS. Macrophages treated with BG45 and HG + LPS were analysed for the expression of pro-IL-1ß, mature IL-1ß, oxidative stress and pro-inflammatory (M1) and anti-inflammatory (M2) factors. KEY FINDINGS: HDAC3 was found to be upregulated in impaired diabetic mice wounds and in macrophages stimulated with HG + LPS. Topical application of BG45 loaded gel accelerated the wound healing in diabetic mice and was evident by improved expression of Collagen-1A, IL-10, TGF-ß, and angiogenesis (CD31, VEGF). BG45 treatment decreased the expression of IL-1ß, TNF-α, and IL-6 (M1 phenotype), reduced oxidative stress and promoted the expression of Arginase-1 and YM1/2 (M2 phenotype) in macrophages treated with HG + LPS. BG45 also improved the expression of CD11b+F4/80+CD206+ cells in wound tissues and reduced expression of inflammatory markers. SIGNIFICANCE: HDAC3 is upregulated in diabetic mice wounds and HDAC3 selective inhibitor promotes the wound healing by regulating macrophage activation, angiogenesis and IL-1ß.

Selective inhibition of PKR by C16 accelerates diabetic wound healing by inhibiting NALP3 expression in mice.

OBJECTIVE AND DESIGN: To understand the expression of dsRNA-dependent protein kinase R (PKR) in impaired diabetic wounds, hyperglycemia was induced in C57/BL6 mice with streptozotocin. Murine macrophage cell line, Raw 264.7, stimulated with high glucose and LPS was used to mimic diabetic wound environment in in-vitro. MATERIALS: Macrophages stimulated with HG + LPS, in presence and absence of PKR inhibitor (C16) and wound tissue samples from topically treated mice with C16, were analyzed for the expression of PKR, NALP3, active caspase-1, mature IL-1ß and phosphorylation of PKR and eIF2α. Wounds tissues were also analyzed for inflammatory cell infiltration by immunohistochemistry, angiogenesis by CD31 staining, collagen expression by western blotting, expression of CD206+ macrophages by flow cytometry and wound strength by texture analyzer. RESULTS: PKR and NALP3 were found to be upregulated in macrophages stimulated with HG + LPS as well as in impaired diabetic wounds. PKR inhibition using C16 ameliorated expression of NALP3, caspase-1, IL-1ß and phosphorylation of PKR and eIF2α, in macrophages and also in diabetic wounds. Treatment with C16 promoted the wound healing in diabetic mice by increasing collagen synthesis, reducing infiltration of F4/80+ macrophages and MPO+ neutrophil cells, increased angiogenesis, and increased number of M2 macrophages. CONCLUSION: PKR inhibition using C16 accelerates the wound healing process in diabetic mice by decreasing NALP3-mediated IL-1ß maturation.

HDAC5 RNA interference ameliorates acute renal injury by upregulating KLF2 and inhibiting NALP3 expression in a mouse model of oxalate nephropathy.

Krüppel-like factor 2 (KLF2) and NLR family pyrin domain containing 3 (NALP3) are important regulators of macrophage activation in the context of various pathological conditions. NALP3 also plays an important role in the maturation of IL-1 ß which is central to the pathogenesis of acute oxalate nephropathy. The functional role of KLF2 and regulation of both KLF2 and NALP3 in the pathogenesis of acute oxalate nephropathy is comparably less studied. Here, we explored the regulation of KLF2 and NALP3 by Histone deacetylase 5 (HDAC5) in oxalate crystals stimulated macrophages, and in the pathogenesis of acute oxalate nephropathy in mice. We observed upregulated expression of HDAC5 along with IL-1ß, Caspase1, and NALP3, while the expression of KLF2 was downregulated in stimulated macrophages and in the renal tissue of mice with acute oxalate nephropathy. We formulated chitosan HDAC5 siRNA nanoparticles to deliver the siRNA in in-vitro and in-vivo settings. siHDAC5 treated cells exhibited decreased expression of IL-1ß, and TNF-α in the supernatant, and reduced expression of NALP3, Pro-caspase1, active caspase1, Pro-IL-1ß, and IL-1ß in cell lysate. Concurrently, the expression of KLF2 was upregulated in HDAC5 depleted cells upon stimulation with crystals. Mice treated with siHDAC5 nanoparticles showed protection against renal impairment with improved renal function (plasma BUN and creatinine levels), reduced inflammation (IL-1ß expression), reduced accumulation of neutrophils, reduced tubular injury, reduced acute renal injury markers (KIM-1, NGAL-1), reduced expression of NALP3, Pro-caspase1, active caspase1, Pro-IL-1ß, and IL-1ß. Whereas, the expression of KLF2 was significantly upregulated by depletion of HDAC5 in mice.

HDAC6 inhibition attenuates renal injury by reducing IL-1ß secretion and RIP kinase mediated necroptosis in acute oxalate nephropathy.

BACKGROUND: Pathogenesis of acute kidney injury is driven by necro-inflammation, which is comprised of IL-1ß mediated inflammation and RIP-1 mediated tubular necroptosis. HDAC6 is reported to regulate both inflammation and cell death. In the present study, we explored the role of HDAC6 in the lysosomal exocytosis of IL-1ß and RIP-1 mediated necroptosis in the context of oxalate nephropathy. METHODS: Raw 264.7 macrophages and NRK52E stimulated with oxalate crystals and LPS with or without HDAC6 inhibitor for in vitro experiments. Acute oxalate nephropathy was induced in C57BL/6 mice by injecting sodium oxalate (75 mg/kg). For the drug intervention study, Tubastain A (TSA) was given an hour before injection of sodium oxalate. Mice were sacrificed 24 hrs after the oxalate injection, blood and kidney were harvested. Blood samples were analyzed for BUN and IL-1ß levels. Renal tissues were analyzed for histology, immunohistochemistry, RNA, and protein expression. RESULTS: HDAC6 and IL-1ß upregulated in crystal stimulated macrophages and acute oxalate nephropathy. Pre-treatment of macrophages with TSA reduced IL-1ß in supernatant without affecting the expression of pro-IL-1ß and mature IL-1ß in cell lysate. The effect of TSA on IL-1ß secretion was influenced by tubulin acetylation. Renal epithelial cell NRK52E stimulated with crystals showed upregulation of necroptosis pathway markers and concentration-dependent cell death. TSA inhibited RIP-1, RIP3, and MLKL expression along with p-MLKL in stimulated epithelial cells. TSA treatment of oxalate nephropathy mice showed decreased inflammation and tubular cell death by regulating IL-1ß and necroptosis and reduced renal injury. CONCLUSION: This study highlights the role of HDAC6 in regulating the tubulin-mediated secretion of IL-1ß and RIP kinase mediated necroptosis in acute oxalate nephropathy.

HDAC6 inhibitor accelerates wound healing by inhibiting tubulin mediated IL-1ß secretion in diabetic mice.

Delayed wound healing in diabetes is characterized by sustained activation of inflammasome and increased expression of IL-1ß in macrophages. Identification and validation of novel pathways to regulate IL-1ß expression will provide therapeutic targets for diabetic wounds. Here we report sustained over-expression of histone deacetylase 6 (HDAC6) in wounds of diabetic mice and its role in delayed wound healing. Topical application of HDAC6 inhibitor; Tubastatin A (TSA) gel promoted the wound healing in diabetic mice. TSA hydrogel reduced the infiltration of neutrophils, T-cells and macrophages in the early phase of wound healing. TSA treatment promoted the wound healing by inducing collagen deposition, angiogenesis (CD31) and fibrotic factors (TGF-ß1) in the late phase of healing. Protein analysis of the diabetic wounds treated with TSA showed increased acetylated α-tubulin and decreased levels of mature IL-1ß with no significant effect on the expression of pro-IL-1ß, pro-caspase-1 and active caspase-1. In in vitro assays, macrophages exhibited upregulation of HDAC6, IL-1ß and downregulation of IL-10 upon stimulation with high glucose and LPS. TSA inhibited the IL-1ß secretion and promoted IL-10 in stimulated macrophages with high glucose and LPS. Further investigations showed that TSA inhibits IL-1ß release by inhibiting tubulin dependent lysosomal exocytosis without affecting its transcription and maturation. Nocodazole (known acetylation inhibitor) pre-treatment inhibited TSA effect on IL-1ß secretion in high glucose stimulated macrophages. Overall, our findings indicate that sustained HDAC6 expression in diabetic wounds contributes to impaired healing responses and HDAC6 may represent a new therapeutic target for diabetic wounds.

Preventive effect of berberine against DMBA-induced breast cancer in female Sprague Dawley rats.

Breast cancer is the prime cause for cancer mortality in women worldwide. The importance of diverse natural and dietary agents to reduce the risk of developing breast cancer is well established. Berberine, a natural isoquinoline alkaloid found in many medicinal plants is widely used in traditional Indian and Chinese medicine. Because of its capability to seize the cell cycle and induce apoptosis of numerous malignant cells, berberine has received considerable attention as a potential anticancer agent. In the present study, breast cancer was induced in Sprague Dawley (SD) rats by intragastric administration of 7, 12-dimethylbenz[a]anthracene (DMBA) at a dose of 80mg/kg of body weight. Treatment of berberine (50mg/kg BW) to breast tumor bearing rats was found to be effective against DMBA induced mammary carcinoma. The increased levels of lipid peroxide (malonaldehyde), pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α), enzymatic antioxidants (SOD and CAT), non-enzymatic antioxidants (GSH and vitamin C) and transcription factor NF-κB were decreased significantly by administration of berberine. Furthermore, RT-PCR and western blot analysis showed the down-regulation of NF-κB and PCNA in breast tumors. Histopathological studies validated that berberine is effective against DMBA induced ductal carcinoma & invasive carcinoma. Altogether, these findings demonstrate the preventive role of berberine against DMBA induced mammary carcinoma in SD rats.

Antiproliferative and antioxidant potential of hesperetin against benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice.

Lung cancer is the foremost cause of cancer mortality and is a growing economic burden worldwide. Chemoprevention, employing the use of natural, dietary or synthetic agents has become an appealing strategy to combat the increasing cases of cancers worldwide. The present study was designed to investigate the mechanism-based chemopreventive nature of hesperetin (HSP) against B[a]P induced lung carcinogenesis in Swiss albino mice. We analyzed the chemopreventive potential of HSP by estimating the status of lipid peroxidation (LPO), enzymic (SOD, CAT, GPx, GR, and GST), nonenzymic antioxidants (GSH, Vit C and Vit E), proinflammatory cytokine (TNF-α), western blotting (CYP1A1, PCNA, Nrf2 and NF-κB expression) and histopathology of lung tissues of control and experimental mice. Administration of B[a]P (50 mg/kg, p.o.) resulted in an increase in lung weight, LPO with concomitant decrease in body weight, enzymic (SOD, CAT, GPx, GR, and GST) and non-enzymic (GSH, Vit C and Vit E) antioxidants. Histological examination of lungs revealed severe alveolar and bronchiolar damages in B[a]P-induced mice. Further, elevated levels of TNF-α along with activated expression of NF-κB, PCNA and CYP1A1, and downregulation of Nrf2 was observed in B[a]P intoxicated animals. Pre- and post-treatment with HSP effectively suppressed B[a]P induced lung carcinoma and the associated preneoplastic lesions by alleviating LPO, modulating antioxidants and decreasing the expression of NF-κB, PCNA and CYP1A1. These findings demonstrate that HSP possesses a potential chemopreventive activity against B[a]P induced lung cancer and this is attributed to its free radical scavenging, antioxidant, anti-inflammatory and antiproliferative properties.