Inhibition of TRPC6 suppressed TGFß-induced fibroblast-myofibroblast transdifferentiation in renal interstitial NRK-49F cells.

Renal fibrosis is a global health concern with limited curative treatment. Canonical transient receptor potential channel 6 (TRPC6), a nonselective cation channel, has been shown to regulate the renal fibrosis in murine models. However, the molecular mechanism is unclear. Fibroblast-myofibroblast transdifferentiation is one of the critical steps in the progression of renal fibrosis. In the present study, we demonstrate that transforming growth factor (TGF)-ß1 exposure significantly increases the TRPC6 expression in renal interstitial fibroblast NRK-49F cells. Pharmacological inhibition of TRPC6 and knockdown of Trpc6 by siRNA alleviate TGF-ß1-increased expression levels of α-smooth muscle actin (α-SMA) and collagen I, two key markers of myofibroblasts. Although direct activation of TRPC6 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) does not affect the expression of α-SMA and collagen I, OAG potentiates TGF-ß1-induced fibroblast-myofibroblast transdifferentiation. Further study demonstrates that TGF-ß1 exposure increases the phosphorylation level of p38 and Yes-associated protein (YAP) translocation into the nuclei. Inhibition of p38 and YAP decreases TGF-ß1-enhanced TRPC6 and α-SMA expression. In conclusion, we demonstrate that TRPC6 is a key regulator of TGF-ß1-induced fibroblast-myofibroblast transdifferentiation and provides the mechanism of how TGF-ß1 exposure regulates TRPC6 expression in NRK-49F fibroblasts.

Suppression of pancreatic beta cell apoptosis by Danzhi Jiangtang capsule contributes to the attenuation of type 1 diabetes in rats.

BACKGROUND: Danzhi Jiangtang Capsule (DJC), a Chinese medicinal formula, has been clinically used for treatment of diabetes for many years. Previous studies have demonstrated that DJC was able to improve pancreatic islet function in diabetes, but the underlying mechanisms remained unclear. METHODS: Streptozotocin (STZ) induced type 1 diabetic rats were treated with DJC for 6 weeks. Fasting plasma insulin and fasting plasma glucose were determined at the end of experiment. Antioxidant status was evaluated by measuring total antioxidant capacity, superoxide dismutase activity and malondialdehyde content in plasma and pancreas. Paraffin sections of pancreas were subjected to H&E staining, TUNEL staining and immunohistochemical examination. Protein levels of Bcl-2, Bax and pancreatic duodenal homeobox-1 (PDX-1) were measured by western blot analysis. Activities of Caspase-3 and Caspase-9 were determined with commercially available kits. RESULTS: Supplementation with DJC resulted in a significant amelioration of type 1 diabetes as manifested by reduced blood glucose, increased fasting plasma insulin and improved body weight gains. The atrophy and reduction of pancreatic islets were also alleviated in DJC supplemented groups. DJC markedly reduced pancreatic beta cell apoptosis, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. The activities of caspase-3 and caspase-9 in pancreas were decreased evidently by DJC treatment. DJC effectively ameliorated oxidative stress in type 1 diabetic rats, with the expression of PDX-1 protein increased markedly. CONCLUSIONS: DJC was capable of attenuating STZ induced type 1 diabetes in rats, which might be attributed to the suppression of pancreatic beta cell apoptosis. This study would provide further evidence for clinical use of DJC in the management of diabetes.

[Sesamin Preconditioning Attenuates Myocardial Ischemia Reperfusion Injury in Rats Through Activation of Akt/eNOS Signaling Pathway].

Objective: To investigate the protective effect of sesamin on myocardial ischemia reperfusion injury in rats, and to study the possible mechanism. Methods: 50 SD rats were randomly divided into control group, sham operated group, model group, high-dose sesamin group( 160 mg / kg) and low-dose sesamin group( 80 mg / kg),with 10 rats in each group. Rats in sesamin groups were administered intragastrically with sesamin for 7 d. Then all rats except those in sham operated group were subjected to myocardial ischemia-myocardial ischemia reperfusion injury model by coronary artery ligation for 40 min and subsequent reperfusion for 120 min. Serum cardiac troponin Ⅰ( c TnⅠ) and lactate dehydrogenase( LDH),levels of total antioxidant capacity( TAOC) and nitric oxide( NO) in serum and myocardial tissues,Caspase-3 activity in myocardial tissues were detected by colorimetric assay. Cardiomyocyte apoptosis was evaluated by TUNEL assay. Phosphorylation level of endothelial nitric oxide synthase( eNOS) and Protein kinase B( Akt), protein expression of superoxide dismutase( SOD) in cardiac tissue were determined by Western blot. Results: Pretreatment with sesamin significantly ameliorated myocardial injury in rats which induced myocardial ischemia and reperfusion injury by reduced levels of serum c TnⅠand LDH( P <0. 05 or P < 0. 01). Supplementation with sesamin resulted in a significant increasing of total antioxidant capacity and NO level in serum and myocardial tissues and cardiomyocyte apoptosis( P < 0. 05 or P < 0. 01),and remarkable decrease the Caspase-3 activity in myocardial tissues and cardiomyocyte apoptosis( P < 0. 05 or P < 0. 01). Sesamin significantly up-regulated the protein expression of SOD in cardiac tissues, and the levels of phosphorylated eNOS and Akt increased notably( P < 0. 05 or P < 0. 01). Conclusion: Pretreatment with sesamin effectively ameliorated myocardial ischemia reperfusion injury in rats, and the mechanism might be related to enhancing its antioxidant capacity and the activation of Akt / eNOS signaling pathway and subsequent increase of NO synthesis and suppression of cardiac myocyte apoptosis.

[The effect of Salvianolic Acid B on the Apoptosis of HUVECs Induced by Intermittent High Glucose].

Objective: To investigate the effect of Salvianolic acid B (Sal B) on the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by intermittent high glucose and to explore the possible mechanisms. Methods: HUVECs were preincubated with Sal B for 24 h, followed by incubation with intermittent high glucose (IHG, 5.5 mmol/L 12 h, 33.3 mmol/L 12 h) for 72 h. The viability of the HUVECs was determined by MTT assay, and the cells apoptosis was measured flow cytometry, respectively. The levels of nitric oxide (NO), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and Caspase-3 activity were determined by colorimetric method. Intracellular ROS was evaluated by fluorescent microscopy. The protein levels of NOX4, p-eNOS, BAX, and BCL-2 were determined by Western-blot. Results: Pretreatment with Sal B significantly ameliorated IHG-induced cells injury as was manifested by increased cell viability, up-regulated eNOS activation, and promoted the release of NO in HUVECs (P < 0.05 or P < 0.01). Sal B evidently suppressed IHG-induced cell apoptosis, down-regulated the expression of BAX protein and up-regulated the expression of BCL-2 protein. The activity of Capase-3 was also significantly reduced. Pre-incubation with Sal B led to a significant enhancement of antioxidant capacity and a reduction of NOX4 protein expression, accompanied by a remarkable decrease of intracellular ROS and MDA content (P < 0.05 or P < 0.01). Conclusion: Sal B is capable of suppressing IHG-induced injury and apoptosis in HUVECs, which might be attributed to the attenuation of oxidative stress, regulation of BCL-2/BAX protein expression, and subsequent suppression of Caspase-3 activity.

[Effects of crocetin on VCAM-1 expression in human umbilical vein endothelial cells and monocyte-endothelial cell adhesion].

Crocetin, a naturally occurring carotenoid, possesses antioxidant and antiatherosclerotic properties, of which the underlying mechanism remains unclear. In the present study, we examined the effects of crocetin (0.1, 1, 10 µmol·L(-1)) on angiotensin II (Ang II, 0.1 µmol·L(-1)) induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and monocyte-endothelial cell adhesion. The effects of crocetin on the activation of nuclear factor kappa B (NF-κB) and intracellular reactive oxygen species (ROS) were also observed. The results demonstrated that crocetin notably suppressed Ang II induced NF-κB activation (P<0.01) and VCAM-1 expression (P<0.05, P<0.01) in HUVECs, accompanied by a markedly reduced monocyte-endothelial cell adhesion (P<0.05, P<0.01). In addition, preincubation with crocetin resulted in a significant enhancement of cellular antioxidant capacity (P<0.05, P<0.01), while Ang II induced intracellular ROS decreased markedly (P<0.05, P<0.01). These results indicated that crocetin was capable of suppressing Ang II induced VCAM-1 expression and monocyte-endothelial cell adhesion by suppression of NF-κB activation, which might be derived from the enhancement of antioxidant capacity and subsequent reduction of intracellular ROS.

[Effect of Danzhi Jiangtang Capsule on Myocardial Fibrosis in Diabetic Rats].

OBJECTIVE: To investigate the effect of Danzhi Jiangtang Capsule on myocardial fibrosis in diabetic rats with fluctuated blood glucose and the possible mechanisms implicated. METHODS: Following induction of diabetes with intraperitoneal injection of streptozotocin (STZ), rats were administered with insulin or glucose at different time during a day to induce blood glucose fluctuation. After treatment with Danzhi Jiangtang Capsule for six weeks, rats were sacrificed and the hearts were collected for the determination of cardiac mass index. Cardiac levels of angiotensin II (Ang II), type I and type III collagens and transforming growth factor-ß1 (TGF-ß1) were assayed by ELISA. Levels of Smad3 phosphorylation and protein expression of matrix metalloproteinase-2 ( MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were determined by Western blot analysis. Total antioxidant capacity and malondialdehyde (MDA) content in cardiac tissues were measured colorimetrically. RESULTS: Treatment with Danzhi Jiangtang Capsule for six weeks significantly reduced cardiac mass index and cardiac levels of type I and type III collagens (P < 0.05 or P < 0.01). Levels of Ang II, TGF-ß1 and Smad3 phosphorylation in cardiac tissues were also decreased markedly (P < 0.05 or P < 0.01). Supplementation with Danzhi Jiangtang Capsule resulted in an evident up-regulation of MMP-2 protein and down-regulation of TIMP-2 protein expression in cardiac tissues (P < 0.05 or P < 0.01). In addition, Danzhi Jiangtang Capsule significantly enhanced total antioxidant capacity in diabetic rats, while cardiac content of MDA was decreased markedly( P < 0.05 or P < 0.01). CONCLUSION: Danzhi Jiangtang Capsule significantly ameliorated myocardial fibrosis in diabetic rats with fluctuated blood glucose, which might be derived from enhancement of antioxidant capacity, suppression of RAS and TGF-ß1/Smad3 signaling pathway and regulation of MMP-2/TIMP-2 protein expression.

[Effect of Serum Containing Sesamin on Angiotensin II-Induced Apoptosis in Rat Cardiomyocytes].

OBJECTIVE: To investigate the effect of serum containing sesamin on angiotension II (Ang II)-induced apoptosis in rat cardiomyocytes and the possible mechanisms. METHODS: H9c2 rat cardiomyocytes were preincubated with serum containing sesamin or blank serum for 12 h, followed by incubation with Ang II for 24 h. Cell viability was assessed by MTT assay and cell apoptosis was evaluated by flow cytometric analysis. Protein expression of BCL-2, BAX, Caspase-3, p47phox and superoxide dismutase (SOD) was determined by Western blot analysis. Levels of intracellular reactive oxygen species (ROS), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) were measured colorimetrically. RESULTS: Preincubation with serum containing sesamin significantly improved cell viability and suppressed cell apoptosis in H9c2 rat cardiomyocytes exposed to Ang II (P < 0.05 or P < 0.01), with the expression of BAX, Caspase-3 and p47phox protein down-regulated and BCL-2 and SOD protein up-regulated markedly (P < 0.05 or P < 0.01). The levels of T-AOC were effectively increased in serum containing sesamin groups, while the levels of intracellular ROS and MDA contents were decreased significantly (P < 0.05 or P < 0.01). Control serum had no influence on the above mentioned measurements. CONCLUSION: Sesamin is capable of suppressing Ang II-induced apoptosis in H9c2 rat cardiomyocytes, which might be derived, at least partly, from amelioration of oxidative stress, regulation of BAX/BCL-2 protein expression and suppression of Caspase-3 protein expression.

Arbidol attenuates liver fibrosis and activation of hepatic stellate cells by blocking TGF-ß1 signaling.

Chronic liver diseases (CLD) impact over 800 million people globally, causing about 2 million deaths annually. Arbidol (ARB), an indole-derivative used to treat influenza virus infection, was extensively used during COVID-19 pandemic in China. In recent years, studies have shown that ARB, compared to other antiviral drugs, exhibits greater liver-protective efficacy, indicating a potential hepatoprotective effect beyond its antiviral activity. However, the mechanism remains unclear. In this study, we investigated the impact of ARB on liver injury/fibrosis in bile duct ligated (BDL) mice and its effect on spontaneous and transforming growth factor ß1 (TGF-ß1)-induced activation of primary cultured hepatic stellate cells (HSCs). Oral administration of ARB significantly ameliorated BDL-induced liver injury/fibrosis as reflected by decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced collagen deposition, and diminished mRNA expression of fibrosis markers. ARB notably inhibited spontaneous and TGF-ß1-induced activation of primary cultured HSCs. Moreover, ARB also drastically attenuated mRNA expression levels of platelet-derived growth factor receptor (Pdgfr), transforming growth factor-beta receptor (Tgfbr) 1, Tgfbr2, matrix metalloproteinase (Mmp)-2, and Mmp-9 in activated HSCs. We further demonstrate that ARB mitigated Smad2/3 phosphorylation in both TGF-ß1 treated HSCs and BDL mice. These data together demonstrate that the therapeutic efficacy of ARB on liver fibrosis is independent of its antiviral activity and likely is achieved by blocking TGF-ß1 signaling-mediated HSC activation.

Discovery of pyridazinone derivatives bearing tetrahydroimidazo[1,2-a]pyrazine scaffold as potent inhibitors of transient receptor potential canonical 5 to ameliorate hypertension-induced renal injury in rats.

Transient receptor potential canonical 5 (TRPC5) is a calcium-permeable non-selective cation channel involved in various pathophysiological processes, including renal injury. Recently, GFB-887, an investigational pyridazinone TRPC5 inhibitor, demonstrated significant therapeutic potential in a Phase II clinical trial for focal segmental glomerulosclerosis (FSGS), a rare and severe form of chronic kidney disease (CKD). In the current study, based on the structure of GFB-887, we conducted extensive structural modification to explore novel TRPC5 inhibitors with desirable drug-like properties and robust nephroprotective efficacy. A series of pyridazinone derivatives featuring a novel tetrahydroimidazo[1,2-a]pyrazine scaffold were synthesized and their activities were evaluated in HEK-293 cells stably expressing TRPC5 using a fluorescence-based Ca2+ mobilization assay. Among these compounds, compound 12 is turned out to be a potent TRPC5 inhibitor with apparent affinity comparable to the parent compound GBF-887. Compound 12 is highly selective on TRPC4/5 over TRPC3/6/7 and hERG channels, along with acceptable pharmacokinetic properties and a favorable safety profile. More importantly, in a rat model of hypertension-induced renal injury, oral administration of compound 12 (10 mg/kg, BID) efficaciously reduced mean blood pressure, inhibited proteinuria, and protected podocyte damage. These findings further confirmed the potential of TRPC5 inhibitors on the CKD treatment and provided compound 12 to be a valuable tool for exploring TRPC4/5 pathophysiology.

Scutellarein attenuates atopic dermatitis by selectively inhibiting transient receptor potential vanilloid 3 channels.

BACKGROUND AND PURPOSE: Atopic dermatitis (AD) is one of the most common chronic inflammatory cutaneous diseases with unmet clinical needs. As a common ingredient found in several medicinal herbs with efficacy on cutaneous inflammatory diseases, Scutellarein (Scu) has been shown to possess anti-inflammatory and anti-proliferative activities. We aimed to evaluate the therapeutic efficacy of Scu against AD and its underlying molecular mechanism. EXPERIMENTAL APPROACH: Efficacy of Scu on AD was evaluated in 2,4-dinitrofluorobenzene (DNFB) and carvacrol-induced dermatitis mouse models. Cytokine mRNA and serum IgE levels were examined using qPCR and ELISA, respectively. Voltage clamp recordings were used to measure currents mediated by transient receptor potential (TRP) channels. In silico docking, site-direct mutagenesis, and covalent modification were used to explore the binding pocket of Scu on TRPV3. KEY RESULTS: Subcutaneous administration of Scu efficaciously suppresses DNFB and carvacrol-induced pruritus, epidermal hyperplasia and skin inflammation in wild type mice but has no additional benefit in Trpv3 knockout mice in the carvacrol model. Scu is a potent and selective TRPV3 channel allosteric negative modulator with an apparent affinity of 1.18 µM. Molecular docking coupled with site-direct mutagenesis and covalent modification of incorporated cysteine residues demonstrate that Scu targets the cavity formed between the pore helix and transmembrane helix S6. Moreover, Scu attenuates endogenous TRPV3 activity in human keratinocytes and inhibits carvacrol-induced proliferative and proinflammatory responses. CONCLUSION AND IMPLICATIONS: Collectively, these data demonstrate that Scu ameliorates carvacrol-induced skin inflammation by directly inhibiting TRPV3, and TRPV3 represents a viable therapeutic target for AD treatment.

Salvianolic acid B inhibits intermittent high glucose-induced INS-1 cell apoptosis through regulation of Bcl-2 proteins and mitochondrial membrane potential.

Blood glucose fluctuations, also referred to as intermittent high glucose, have been validated to be more harmful than sustained high glucose in exacerbating pancreatic dysfunction by inducing ß cell apoptosis. Salvianolic acid B (Sal B), an aqueous component of Salvia miltiorrhiza, has been proved beneficial to pancreatic islet function in diabetes, but the underlying mechanisms remain to be elucidated. The present study investigated the protective effect of Sal B on INS-1 cells exposed to intermittent high glucose and the possible mechanisms implicated. The results indicated that Sal B was able to restore cell viability and suppress INS-1 cell apoptosis induced by intermittent high glucose. Preincubation with Sal B led to a significant decrease of caspase-9 and caspase-3 activity and poly ADP-ribose polymerase (PARP) cleavage. Exposure to intermittent high glucose induced significant up-regulation of proapoptotic proteins, down-regulation of antiapoptotic protein and depolarization of mitochondrial membrane potential (MMP) in INS-1 cells, while these changes were reversed effectively in Sal B treated groups. In addition, Sal B markedly attenuated intermittent high glucose-induced oxidative stress as manifested by notably decreased levels of intracellular reactive oxygen species and malondialdehyde (MDA). Taken together, these results indicate that Sal B is able to suppress intermittent high glucose-induced INS-1 cell apoptosis, which might be ascribed to regulation of Bcl-2 family protein expression and preservation of mitochondrial membrane potential.

Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis.

Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

Sesamin suppresses STZ induced INS-1 cell apoptosis through inhibition of NF-κB activation and regulation of Bcl-2 family protein expression.

Diverse risk factors for diabetes can induce oxidative stress, leading to pancreatic beta cell damage and insulin secretion dysfunction. In the present study, we evaluated the effect of sesamin on streptozotocin (STZ) induced apoptosis in INS-1 cells and the possible mechanisms implicated. After preincubation with indicated concentrations of sesamin (0.1, 1.0 and 10.0µmol/l) for 24h, INS-1 cells were exposed to STZ (3mmol/l) for 12h. Sesamin effectively improved STZ induced cell damage as determined by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] assay and insulin secretion capacity, and suppressed STZ induced cell apoptosis as evaluated by flow cytometry using annexin V and propidium iodide double staining. Western blot analysis demonstrated that sesamin markedly suppressed STZ induced nuclear factor kappa B (NF-κB) activation, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. Preincubation with sesamin resulted in an evident enhancement of total antioxidant capacity in INS-1 cells, accompanied by a significant reduction of intracellular reactive oxygen species and malondialdehyde, an end product of lipid peroxidation. Taken together, these findings suggested that sesamin was capable of suppressing STZ induced INS-1 cell apoptosis, which might be ascribed, at least partly, to the inhibition of NF-κB activation and subsequent regulation of Bcl-2 family protein expression. This study would provide a potential target for treatment of diabetes with sesamin as well as other antioxidants.

Suppression of TGF-ß1/Smad signaling pathway by sesamin contributes to the attenuation of myocardial fibrosis in spontaneously hypertensive rats.

This study investigated the effect of sesamin on myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the possible mechanisms involved. Twenty-eight male SHRs were randomly allocated to SHR group, Ses160 group (sesamin 160 mg/kg), Ses80 group (sesamin 80 mg/kg) and Cap30 group (captopril 30 mg/kg). Seven male WKY rats were used as control. Sesamin and captopril were administered intragastrically for 12 weeks. Captopril significantly reduced systolic blood pressure and angiotensin II (Ang II) levels in SHRs, accompanied by a marked attenuation of left ventricular hypertrophy (LVH) and collagen deposition (P <0.05 or P <0.01). Though sesamin had no significant influence on Ang II levels, and the hypotensive effect was also significantly inferior to that of captopril (P <0.05 or P <0.01), however, the improvement of LVH and collagen deposition was similar to that in captopril group. Sesamin markedly reduced transforming growth factor-ß1 (TGF-ß1) content in cardiac tissues, with Smad3 phosphorylation decreased and Smad7 protein expression increased notably (P <0.05 or P <0.01). Protein expression of type I collagen and type III collagen, target genes of Smad3, was down-regulated markedly by sesamin (P <0.05 or P <0.01). In addition, sesamin significantly increased total antioxidant capacity and superoxide dismutase protein in cardiac tissues (P <0.05 or P <0.01), while the expression of NADPH oxidase subunit p47phox and malondialdehyde content were reduced markedly (P <0.05 or P <0.01). In vitro studies also demonstrated that sesamin was able to suppress Ang II induced phosphorylation of Smad3 and secretion of TGF-ß1 and type I and type III collagen in cultured rat cardiac fibroblasts. These data suggest that sesamin is capable of attenuating hypertensive myocardial fibrosis through, at least partly, suppression of TGF-ß1/Smad signaling pathway.