Cannabinoid receptor 2 activation alleviates diabetes-induced cardiac dysfunction, inflammation, oxidative stress, and fibrosis.

Diabetes mellitus promotes accelerated cardiovascular aging and inflammation, which in turn facilitate the development of cardiomyopathy/heart failure. High glucose-induced oxidative/nitrative stress, activation of various pro-inflammatory, and cell death pathways are critical in the initiation and progression of the changes culminating in diabetic cardiomyopathy. Cannabinoid 2 receptor (CB2R) activation in inflammatory cells and activated endothelium attenuates the pathological changes associated with atherosclerosis, myocardial infarction, stroke, and hepatic cardiomyopathy. In this study, we explored the role of CB2R signaling in myocardial dysfunction, oxidative/nitrative stress, inflammation, cell death, remodeling, and fibrosis associated with diabetic cardiomyopathy in type 1 diabetic mice. Control human heart left ventricles and atrial appendages, similarly to mouse hearts, had negligible CB2R expression determine by RNA sequencing or real-time RT-PCR. Diabetic cardiomyopathy was characterized by impaired diastolic and systolic cardiac function, enhanced myocardial CB2R expression, oxidative/nitrative stress, and pro-inflammatory response (tumor necrosis factor-α, interleukin-1ß, intracellular adhesion molecule 1, macrophage inflammatory protein-1, monocyte chemoattractant protein-1), macrophage infiltration, fibrosis, and cell death. Pharmacological activation of CB2R with a selective agonist attenuated diabetes-induced inflammation, oxidative/nitrative stress, fibrosis and cell demise, and consequent cardiac dysfunction without affecting hyperglycemia. In contrast, genetic deletion of CB2R aggravated myocardial pathology. Thus, selective activation of CB2R ameliorates diabetes-induced myocardial tissue injury and preserves the functional contractile capacity of the myocardium in the diabetic milieu. This is particularly encouraging, since unlike CB1R agonists, CB2R agonists do not elicit psychoactive activity and cardiovascular side effects and are potential clinical candidates in the treatment of diabetic cardiovascular and other complications.

Potential role of genipin in cancer therapy.

Genipin, an aglycone derived from the iridoid glycoside, geniposide, is isolated and characterized from the extract of Gardenia jasminoides Ellis fruit (family Rubiaceae). It has long been used in traditional oriental medicine for the prevention and treatment of several inflammation driven diseases, including cancer. Genipin has been shown to have hepatoprotective activity acting as a potent antioxidant and inhibitor of mitochondrial uncoupling protein 2 (UCP2), and also reported to exert significant anticancer effects. It is an excellent crosslinking agent that helps to make novel sustained or delayed release nanoparticle formulations. In this review, we present the latest developments of genipin as an anticancer agent and briefly describe its diverse mechanism(s) of action. Several lines of evidence suggest that genipin is a potent inhibitor of UCP2, which functions as a tumor promoter in a variety of cancers, attenuates generation of reactive oxygen species and the expression of matrix metalloproteinase 2, as well as induces caspase-dependent apoptosis in vitro and in in vivo models. These finding suggests that genipin can serve as both a prominent anticancer agent as well as a potent crosslinking drug that may find useful application in several novel pharmaceutical formulations.

Fisetin Confers Cardioprotection against Myocardial Ischemia Reperfusion Injury by Suppressing Mitochondrial Oxidative Stress and Mitochondrial Dysfunction and Inhibiting Glycogen Synthase Kinase 3ß Activity.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide. Timely reperfusion is considered an optimal treatment for AMI. Paradoxically, the procedure of reperfusion can itself cause myocardial tissue injury. Therefore, a strategy to minimize the reperfusion-induced myocardial tissue injury is vital for salvaging the healthy myocardium. Herein, we investigated the cardioprotective effects of fisetin, a natural flavonoid, against ischemia/reperfusion (I/R) injury (IRI) using a Langendorff isolated heart perfusion system. I/R produced significant myocardial tissue injury, which was characterized by elevated levels of lactate dehydrogenase and creatine kinase in the perfusate and decreased indices of hemodynamic parameters. Furthermore, I/R resulted in elevated oxidative stress, uncoupling of the mitochondrial electron transport chain, increased mitochondrial swelling, a decrease of the mitochondrial membrane potential, and induction of apoptosis. Moreover, IRI was associated with a loss of the mitochondrial structure and decreased mitochondrial biogenesis. However, when the animals were pretreated with fisetin, it significantly attenuated the I/R-induced myocardial tissue injury, blunted the oxidative stress, and restored the structure and function of mitochondria. Mechanistically, the fisetin effects were found to be mediated via inhibition of glycogen synthase kinase 3ß (GSK3ß), which was confirmed by a biochemical assay and molecular docking studies.

Protection by dimethyl fumarate against diabetic cardiomyopathy in type 1 diabetic mice likely via activation of nuclear factor erythroid-2 related factor 2.

Oxidative stress and inflammation play key roles in the development of diabetic cardiomyopathy (DCM). Dimethyl fumarate (DMF), an FDA approved medicine for relapsing multiple sclerosis, has manifested its antioxidant and anti-inflammatory function mostly in the central nervous system. In this study, we investigated whether DMF could attenuate the development of DCM. Type 1 diabetes mouse model was established using multiple low-dose streptozotocin, and the diabetic mice were treated with DMF (10 mg/kg body weight) for 3 months. Cardiac functions were determined using echocardiography. Oxidative stress, pro-inflammatory cytokines and pro-fibrotic markers were determined with commercially available kits, real-time quantitative PCR or western blot techniques. DCM was characterized by diminished cardiac function, accompanied by oxidative stress and enhanced expression of pro-inflammatory cytokines. Diabetic cardiac tissue exhibited marked fibrosis, revealed by extracellular matrix deposition as determined by Sirius red staining of the myocardial tissues. Furthermore, Nrf2 and its downstream effectors were repressed in diabetic myocardium. On the contrary, diabetic animals treated with DMF exhibited blunted oxidative stress, inflammation, fibrosis and this correlated with Nrf2 activation. Our findings suggest that DMF could potentially thwart diabetes-induced myocardial tissue injury, likely via activation of Nrf2 function, providing firm impetus for future repurposing of DMF in the management of DCM.

Nootkatone confers hepatoprotective and anti-fibrotic actions in a murine model of liver fibrosis by suppressing oxidative stress, inflammation, and apoptosis.

In this study, the hepatoprotective and anti-fibrotic actions of nootkatone (NTK) were investigated using carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. CCl4 administration elevated serum aspartate and alanine transaminases levels, respectively. In addition, CCl4 produced hepatic oxidative and nitrative stress, characterized by diminished hemeoxygenase-1 expression, antioxidant defenses, and accumulation of 4-hydroxynonenal and 3-nitrotyrosine. Furthermore, CCl4 administration evoked profound expression of pro-inflammatory cytokine expressions such as tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-1ß in hepatic tissues, which corroborated with nuclear factor κB activation. Additionally, CCl4 -treated animals exhibited higher apoptosis, characterized by increased caspase 3 activity, DNA fragmentation, and poly (ADP-ribose) polymerase activation. Moreover, histological and biochemical investigations revealed marked fibrosis in the livers of CCl4 -administered animals. However, NTK treatment mitigated CCl4 -induced phenotypic changes. In conclusion, our findings suggest that NTK exerts hepatoprotective and anti-fibrotic actions by suppressing oxidative stress, inflammation, and apoptosis.

Genipin attenuates cisplatin-induced nephrotoxicity by counteracting oxidative stress, inflammation, and apoptosis.

Cisplatin (CP) is a potent and widely used chemotherapeutic agent. However, the clinical benefits of CP are compromised because it elicits nephrotoxicity and ototoxicity. In this study, we investigated the nephroprotective effects of the phytochemical genipin (GP) isolated from the gardenia (Gardenia jasminoides) fruit, using a murine model of CP-induced nephropathy. GP pretreatment attenuated the CP-induced renal tissue injury by diminishing the serum blood urea nitrogen, creatinine, and cystatin C levels, as well as those of kidney injury molecule-1. In addition, GP attenuated the CP-induced oxidative/nitrative stress by suppressing the activation of NADPH oxidase, augmenting the endogenous antioxidant defense system, and diminishing the accumulation of 4-hydroxynonenal and 3-nitrotyrosine in renal tissues. Furthermore, reduced levels of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta indicated that CP-induced renal inflammation was mitigated upon the treatment with GP. GP also attenuated the CP-induced activation of mitogen-activated protein kinases, excessive activities of caspase-3/7 and poly(ADP-ribose) polymerase, DNA fragmentation, and apoptosis. When administered 12h after the onset of kidney injury, GP showed a therapeutic effect by ameliorating CP-induced nephrotoxicity. Moreover, GP synergistically enhanced the CP-induced cell death of T24 human bladder cancer cells. Collectively, our data indicate that GP attenuated the CP-induced renal tissue injury by abrogating oxidative/nitrative stress and inflammation and by blocking cell death pathways, thereby improving the renal function. Thus, our results suggest that the use of GP may be a promising new protective strategy against cisplatin-induced nephrotoxicity.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Plant-Derived Agents for Counteracting Cisplatin-Induced Nephrotoxicity.

Cisplatin (CSP) is a chemotherapeutic agent commonly used to treat a variety of malignancies. The major setback with CSP treatment is that its clinical efficacy is compromised by its induction of organ toxicity, particular to the kidneys and ears. Despite the significant strides that have been made in understanding the mechanisms underlying CSP-induced renal toxicity, advances in developing renoprotective strategies are still lacking. In addition, the renoprotective approaches described in the literature reveal partial amelioration of CSP-induced renal toxicity, stressing the need to develop potent combinatorial/synergistic agents for the mitigation of renal toxicity. However, the ideal renoprotective adjuvant should not interfere with the anticancer efficacy of CSP. In this review, we have discussed the progress made in utilizing plant-derived agents (phytochemicals) to combat CSP-induced nephrotoxicity in preclinical studies. Furthermore, we have also presented strategies to utilize phytochemicals as prototypes for the development of novel renoprotective agents for counteracting chemotherapy-induced renal damage.

The Role of Oxidative Stress in Myocardial Ischemia and Reperfusion Injury and Remodeling: Revisited.

Oxidative and reductive stress are dual dynamic phases experienced by the cells undergoing adaptation towards endogenous or exogenous noxious stimulus. The former arises due to the imbalance between the reactive oxygen species production and antioxidant defenses, while the latter is due to the aberrant increase in the reducing equivalents. Mitochondrial malfunction is the common denominator arising from the aberrant functioning of the rheostat that maintains the homeostasis between oxidative and reductive stress. Recent experimental evidences suggest that the maladaptation during oxidative stress could play a pivotal role in the pathophysiology of major cardiovascular diseases such as myocardial infraction, atherosclerosis, and diabetic cardiovascular complications. In this review we have discussed the role of oxidative and reductive stress pathways in the pathogenesis of myocardial ischemia/reperfusion injury and diabetic cardiomyopathy (DCM). Furthermore, we have provided impetus for the development of subcellular organelle targeted antioxidant drug therapy for thwarting the deterioration of the failing myocardium in the aforementioned cardiovascular conditions.

Cardioprotective Potentials of Plant-Derived Small Molecules against Doxorubicin Associated Cardiotoxicity.

Doxorubicin (DOX) is a potent and widely used anthracycline antibiotic for the treatment of several malignancies. Unfortunately, the clinical utility of DOX is often restricted due to the elicitation of organ toxicity. Particularly, the increased risk for the development of dilated cardiomyopathy by DOX among the cancer survivors warrants major attention from the physicians as well as researchers to develop adjuvant agents to neutralize the noxious effects of DOX on the healthy myocardium. Despite these pitfalls, the use of traditional cytotoxic drugs continues to be the mainstay treatment for several types of cancer. Recently, phytochemicals have gained attention for their anticancer, chemopreventive, and cardioprotective activities. The ideal cardioprotective agents should not compromise the clinical efficacy of DOX and should be devoid of cumulative or irreversible toxicity on the naïve tissues. Furthermore, adjuvants possessing synergistic anticancer activity and quelling of chemoresistance would significantly enhance the clinical utility in combating DOX-induced cardiotoxicity. The present review renders an overview of cardioprotective effects of plant-derived small molecules and their purported mechanisms against DOX-induced cardiotoxicity. Phytochemicals serve as the reservoirs of pharmacophore which can be utilized as templates for developing safe and potential novel cardioprotective agents in combating DOX-induced cardiotoxicity.

Phytochemicals as Prototypes for Pharmaceutical Leads Towards Drug Development Against Diabetic Cardiomyopathy.

Globally diabetes mellitus (DM) is swiftly reaching epidemic proportions and impose major health care and socio-economic challenges that are associated with its complications. DM is considered as the major risk factor for the development of debilitating micro & macro vascular complications. Clinical studies have revealed that development of diabetic cardiomyopathy (DCM) in subjects with diabetes can occur both- dependent and independent of pre-existing increased risk factors such as poor glycemic control, hyperlipidemia, and or hypertension. Therefore, DCM represents as a major challenge for the clinical community for the prompt diagnosis and devising the treatment paradigm to combat the diabetes induced cardiac dysfunction. In Chinese traditional medical practice, heart ailments have been coped with herbal extracts. Phytochemicals bioavailability and pharmacokinetic properties are to yet be established completely in human subjects. However, tremendous progress has been made to isolate, purify the phytochemicals and characterize their effects on mitigating the development of DCM in pre-clinical models. Currently there are no approved drugs available for the treatment of DCM. In this review, we have discussed the progress made in understanding the mechanisms for the phytochemicals cardio-protective actions in the diabetic milieu and their caveats and provide future perspectives for proposing these agents to serve as prototypes in the development of drugs for the management of DCM.

Methylene Blue inhibits the inflammatory process of the acetic acid-induced colitis in the rat colonic mucosa.

Inflammatory bowel disease is a serious health problem. Although it has been widely investigated, treatment of inflammatory bowel diseases currently remains as a challenging clinical problem. Over production of nitric oxide has been demonstrated to cause tissue damage and inflammation. In this study, the effect of methylene blue (MB), a well-known inhibitor of nitric oxide synthesis, was investigated in acetic acid (AA)-induced colitis model in Sprague-Dawley rats. Eighty male rats randomized into 4 groups (control, control MB, colitis, colitis + MB). AA was applied to groups 3 and 4. MB was added into group 2 and 4. Three days later, animals were sacrificed and 8 cm distal colonic segment resected and the specimens are examined using macroscopical, histological, and biochemical methods. The results of the macroscopic and microscopic examination showed that in group 4 the mucosal damage and inflammation score significantly lower than group 3. Increased intestinal permeability in acetic acid-administered group was significantly reversed by MB application. Myeloperoxidase activity and malondialdehyde levels increased significantly, while superoxide dismutase and catalase activities were suppressed after AA-administration. These biochemical parameters were reversed in MB-treated group. Administration of acetic acid resulted in increased levels of tumor necrosis factor-α, interleukin-1ß, interleukin-6, total nitrite/nitrate levels and nitric oxide synthase activity. These biochemical alterations were significantly reversed by MB application also. In conclusion, our results indicate that MB decreases the level of nitric oxide and decreases inflammation in acetic acid-induced colitis.

Effects of cannabidiol on contractions and calcium signaling in rat ventricular myocytes.

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca(2+) was measured in cells loaded with the Ca(2+) sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca(2+) currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1µM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca(2+) transients. However, the amplitudes of caffeine-evoked Ca(2+) transients and the rate of recovery of electrically evoked Ca(2+) transients following caffeine application were not altered. CBD (1µM) significantly decreased the duration of APs. Further studies on L-type Ca(2+) channels indicated that CBD inhibits these channels with IC50 of 0.1µM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [(3)H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca(2+) channels at a site different than dihydropyridine binding site and inhibits excitation-contraction coupling in cardiomyocytes.

Effects of endogenous cannabinoid anandamide on cardiac Na⁺/Ca²âº exchanger.

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na(+)/Ca(2+) exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 µM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 µM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 µM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 µM) and AM630 (1 µM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 µg/ml) treatment or following the inclusion of GDP-ß-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 µM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1.

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.