Silymarin Administration Attenuates Cirrhotic-induced Cardiac Abnormality in the Rats: A Possible Role of ß1-adrenergic Receptors and L-type Voltage-Dependent Calcium Channels.

Background: Cirrhotic cardiomyopathy is a well-recognized cardiac dysfunction in cirrhotic patients. Studies have confirmed the protective effects of silymarin in different types of cardiac injury. This study aimed to examine the effectiveness and molecular mechanism of silymarin against myocardial dysfunction and hypertrophy in a rat model of cirrhosis. Methods: The experiment was performed at Alborz University of Medical Sciences (Karaj, Iran) during 2020-2021. Thirty-two male Wistar rats were randomly divided into four groups of Sham-operated (control group for surgical procedures), Bile Duct Ligated (BDL), and two Silymarin extract (SE)-treated groups of 300 and 600 mg/Kg/day. After 28 days, serum levels of AST, ALT, GGT, and ALP, liver histopathological status, as well as cardiac mechanical function, were assessed. Cardiac ß1-adrenergic receptors (ß1-AR), L-type voltage-dependent calcium channels (L-VDCC), and GATA4 mRNA expression were also determined using real-time RT-PCR. Data analysis was performed using the one-way ANOVA followed by Duncan's multiple range test. Histological data has been analyzed with Kruskal-Wallis nonparametric test. The analysis was performed at P≤0.05. Results: BDL was associated with a significant elevation in serum AST, ALT, GGT, and ALP, development of necrosis and fibrosis of the liver texture, increased Heart Weight and Heart Weight to Body Weight ratio, enhanced cardiac mechanical function as well as a significant up-regulation of ventricular ß1-AR and L-VDCC. Administration of SE600, but not SE300, significantly reduced the serum levels of the enzymes and alleviated signs of liver necrosis and fibrosis. Cirrhotic-induced cardiac dysfunction was also restored by SE600, but not by the lower dose. In addition, cardiac expression of the ß1-AR and L-VDCC was down-regulated toward normal values by either higher or lower doses of the SE. Conclusion: Silymarin treatment in higher dose attenuated cirrhosis-associated cardiac remodeling and reduced cardiac mechanical dysfunctions.

Ischemic Preconditioning Efficacy Following Anabolic Steroid Usage: A Clear Difference Between Sedentary and Exercise-Trained Rat Hearts.

Previous studies show that anabolic steroids impair innate cardioprotective mechanisms. Here, we investigated the effect of supraphysiological doses of nandrolone on ischemic preconditioning (IPC) as a potent cardioprotective tool against ischemia reperfusion (IR) injury in rat hearts. Male Wistar rats in two experimental settings of sedentary and exercise-trained (60 min/day swimming, 5 days/week, for 8 weeks) were either pretreated with intramuscular injections of arachis oil (Arach, n = 16) as vehicle or nandrolone decanoate (ND, n = 8), 10 mg/kg/week, for 8 weeks. At the end, the hearts were excised and perfused in a Langendorff system. Then, the vehicle-treated hearts subdivided into the IR (30 min of LAD coronary artery occlusion and 120 min reperfusion, n = 8) and IPC (three cycles of 3-min ischemia and 3-min reperfusion before test ischemia, n = 8) groups and nandrolone-treated hearts served as ND + IPC (nandrolone pretreatment before IR and IPC protocols, n = 8) group. Post-ischemic cardiac function and infarct size were assessed. Reperfusion arrhythmias were analyzed using a standard scoring system. In sedentary hearts, ND slightly increased heart-to-body weight ratio and increased baseline cardiac contractile function. In trained hearts, ND markedly increased heart-to-body weight ratio which was also associated with enhanced baseline cardiac function. ND pretreatment enhanced protective effects of IPC in sedentary group; however, abolished these effects in exercise-trained group. The arrhythmia score was not significantly different between nandrolone-treated groups vs. respective preconditioned groups. Our findings show that ND impairs IPC-induced cardioprotection in exercise-trained rat hearts. Cardiac hypertrophy seems to play a crucial role in this response.

Hemodynamic Changes in Experimentally Envenomed Anaesthetized Rats by Intravenous Injection of Hemiscorpius lepturus Venom.

BACKGROUND: We investigated the hemodynamic changes (Inotropic, chronotropic and arrhythmogenic) in intravenously envenomed anesthetized rats with Hemiscorpius lepturus venom. The neutralizing potencies of different drugs and commercial antivenom were assessed simultaneously. METHODS: Different doses of the crude venom (100, 200 and 400µg/rat) were injected during five minutes via the femoral vein and cardiovascular changes were recorded in rats in Razi Institute Corporation, Karaj, Iran in 2017. The drugs (Atropine, lidocaine, propranolol and prazosin) were injected before the venom for determination of the counteracting effects. Different volumes (100, 500 and 1000µl) of the antivenom were pre envenomed to neutralize cardiovascular changes. RESULTS: Temporary hypertension and bradycardia with no arrhythmogenic effects were depicted within twenty minutes. There was a difference in arterial pressure between the venom (400µg/rat) and the vehicle at 8 minutes (114.68±5.1mmHg versus 70.2±4.3mmHg). Elevation of the mean arterial pressure was inhibited by propranolol (2 mg/kg) and neutralized by prazosin (1mg/kg) while lidocaine (4mg/kg) and atropine (1mg/kg) had no effects. Premedication with Iranian commercial antivenom (1000µl) produced surprisingly temporary hypertension compared to the vehicle (140.84±4.5 versus 84.3±3.2). It had no neutralizing properties on blood pressure variation before the venom injection. Volume-expanded hypertension phenomenon was ruled out in a parallel study. CONCLUSION: This venom has vasoconstrictive effects in rats probably due to the presence of norepinephrine like materials in its content or liberated from adrenal gland inhibited by prazosin premedication. The neutralizing effects of antivenom on venom-induced hypertension are questionable.

Involvement of neuronal pathways in the protective effects of hindlimb perconditioning during renal ischemia.

Remote ischemic perconditioning (RPEC) is a therapeutic intervention that has been demonstrated to reduce renal ischemia/reperfusion (I/R) injury. However, the underlying renal protective mechanism remains unclear. The present study hypothesized that RPEC may utilize neural pathways to transfer the protective signal from the perconditioned hindlimb to the kidney. Following a right nephrectomy, rats were randomly allocated into five groups (n=6). The sham group underwent the surgical protocol only. In all other groups, the left renal pedicle was clamped for 45 min and reperfused for 24 h. The I/R control group then underwent 45 min ischemia and 24 h reperfusion (I/R) with no more intervention but the I/R-NR control group underwent the ischemia and reperfusion followed by left femoral nerve (FN) and sciatic nerve (SN) resection. The RPEC group underwent ischemia and reperfusion followed by four cycles of 5 min occlusions of the left femoral artery and 5 min reperfusion. Finally, the RPEC-NR group underwent ischemia and reperfusion followed by left FN and SN resection. Following 24 h, renal functional indices, plasma blood urea nitrogen (BUN) and creatinine (Cr) levels, urinary N-acetyl-ß-glucosaminidase (NAG) release and histopathological changes were assessed. Compared with the sham group, ischemia and reperfusion in the sham and I/R control groups resulted in renal dysfunction, indicated by significantly increased levels of BUN and Cr. This was accompanied by increased urinary NAG activity and morphological damage observed in control groups. In the RPEC group, renal histology and function were significantly improved compared with the control groups. However, FN and SN resection eliminated the protection of the kidney, which was induced by RPEC. In conclusion, remote hindlimb ischemic perconditioning reduced renal I/R injury in the rat kidney in a manner that potentially involves a neural pathway.

Cardiotoxic and Arrhythmogenic Effects of Hemiscorpius lepturus Scorpion Venom in Rats.

BACKGROUND: Envenomation by Hemiscorpius lepturus is not painful and the clinical manifestations include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions, cardiac arrhythmia and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants. Cardiac effects of envenomation by H. lepturus venom including inotropic, chronotropic and arrhythmogenic properties are not studied as now in rat hearts with Langendorff apparatus. METHODS: Rat hearts were allowed to equilibrate in its buffer and cardiotropic plus arrhythmogenic effects induced by injection of different doses of H. lepturus venom were detected and recorded by computer acquisition based data in Langendorff apparatus. The neutralizing effects of Razi Institute antivenom and autonomic drugs were assayed in parallel studies. RESULTS: Hemiscorpius lepturus venom (25 µg/100 l) treatment caused a negative inotropic (65.4 ± 3.2 versus 110.2 ± 3.4) and chronotropic effects (186.3 ± 4.2 versus 302 ± 6.3) in comparison to normal saline. Arrhythmogenic aspects including bradycardia, QRS widening and ST depression were induced by venom injection. Pre venom treatment (20 min) of Razi Institute antivenom (10 µl) neutralized cardiotropic effects but post venom injection (15 min later) had no therapeutic role. Pre (10 min before) and post (15 min after) injection of adrenaline (10 µl) neneutralized cardiotropic effects while pre venom injection (20 min) of propanolol (10 µl) had aggravating effects. CONCLUSION: Our study paves the way for further in vivo investigation of cardiovascular effects of this venom for finding suitable treatments instead of its ordinary antivenom medication in cardiogenic shock induced by the venom.

Oxytocin protects cardiomyocytes from apoptosis induced by ischemia-reperfusion in rat heart: role of mitochondrial ATP-dependent potassium channel and permeability transition pore.

The current study examines the protective effect of oxytocin (OT) on cardiomyocyte apoptosis modulated by mitochondrial ATP-dependent potassium (mitoKATP) channel and permeability transition pore (mPTP) in the preconditioned myocardium of anesthetized rats. Eighty rats were equally divided into eight groups. The hearts of all animals except for the sham group were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin, 5-hydroxydeconoate (5-HD), a specific inhibitor of the mitoKATP channel, and atractyloside (ATRC), an mPTP opener, were used prior to ischemia. Hemodynamic parameters were recorded throughout the experiment. Evaluations were made by infarct size, plasma lactate dehydrogenase level (LDH), transmission electron microscopy (TEM) and immunohistochemistry studies. OT prevented mean arterial pressure drop during early phase of ischemia and reperfusion. Treatment with OT before IR induction normalizes cardiomyocytes both in light microscopy and TEM observations. In addition, OT significantly reduced TUNEL- and increased Bcl-2-labeled positive cell number relative to IR (p<0.05). However, 5HD or ATRC inhibited the protective effects of OT on cardiomyocytes damaged by IR (p<0.05). Ultrastructural changes including extensive myofibril loss, sarcolemmal disruption and mitochondrial swelling due to amorphous dens bodies indicate necrosis induction in 5HD and ATRC as well as in IR groups. Restoration of immunohistochemistry parameters and protection against IR-induced ultrastructural changes confirm OT cardioprotective effects via mitoKATP channel and mPTP modulation in apoptosis induced by ischemia-reperfusion.

The anti-infarct, antistunning and antiarrhythmic effects of oleuropein in isolated rat heart.

Previous studies have reported that oleuropein, the major constituent of olive leaves, has cardioprotective effects. There is no report related to oleuropein and ischemic-reperfusion injuries (cardiac dysfunction and myocardial infarction) as well as preconditioning in rat hearts. 56 male Wistar rats were divided into 7 groups (n=8). Group 1 as the control group and groups 2 to 7 as the treatment groups that received a single dose of oleuropein (100 mg/kg, i.p.) 1, 3, 6, 12, 24 and 48 hours before the excision of the heart, respectively. After these times, their hearts were excised and subjected to 30 min regional ischemia and 120 min reperfusion under Langendorff apparatus. Electrocardiogram and intraventricular pressures were monitored and recorded throughout the procedure. Finally, infarct size was measured by triphenyltetrazolium chloride staining. Compared to the control group, oleuropein significantly reduced infarct size and reperfusion-induced cardiac dysfunction in groups 2 and 3. Oleuropein markedly attenuated both ischemic and reperfusion arrhythmias in groups 2 and 3. There was no significant difference between other groups (4 to 7) than the control group. Heart rate had no significant difference among all of the groups. These results indicate that pretreatment of rats with a single dose of intraperitoneal oleuropein could protect their heart against ischemic-reperfusion injury for at least 3 hours. However, it has no preconditioning effect, since oleuropein had not cardioprotective effects 24 hour later.

Hyperoxic preconditioning fails to confer additional protection against ischemia-reperfusion injury in acute diabetic rat heart.

Experimental studies show that detrimental effects of ischemia-reperfusion (I/R) injury can be attenuated by hyperoxic preconditioning in normal hearts, however, there are few studies about hyperoxia effects in diseased myocardium. The present study was designed to assess the cardioprotective effects of hyperoxia pretreatment (≥ 95 % O2) in acute diabetic rat hearts. Normal and one week acute diabetic rats were either exposed to 60 (H60) and 180 (H180) min of hyperoxia or exposed to normal atmospheric air (21 % O2). Then hearts were isolated immediately and subjected to 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size, cardiomyocyte apoptosis, enzymes release and ischemia induced arrhythmias were determined. Heart of diabetic control rats had less infarct size and decreased LDH and CK-MB release compared to normal hearts. 60 and 180 min of hyperoxia reduced myocardial infarct size and enzymes release in normal hearts. 180 min of hyperoxia also decreased cardiomyocytes apoptosis in normal state. On the other hand, protective values of hyperoxia were not significantly different in diabetic hearts. Moreover, hyperoxia reduced severity of ventricular arrhythmias in normal rat hearts whereas; it did not confer any additional antiarrhythmic protection in diabetic hearts. These findings suggest that diabetic hearts are less susceptible to ischemia-induced arrhythmias and infarction. Hyperoxia greatly protects rat hearts against I/R injury in normal hearts, however, it could not provide added cardioprotective effects in acute phase of diabetes.

Ischemia and reperfusion-induced arrhythmias: role of hyperoxic preconditioning.

BACKGROUND: Hyperoxic preconditioning is known to protect the heart against necrosis and contractile dysfunction, but protection against arrhythmias has not been well characterized. OBJECTIVE: The authors hypothesized that pre-exposure to normobaric hyperoxia (H) reduces ischemia and reperfusion-induced arrhythmias in isolated rat hearts. METHODS: Following 60 and 180 min of hyperoxia treatment, rat hearts were isolated immediately (H60 and H180) or 24 h afterward (H60/24 and H180/24), and subjected to 30 min of regional ischemia followed by 120 min of reperfusion. Occurrence, number, and duration of arrhythmias were analyzed during ischemia and reperfusion. In addition, cardiac infarct size was also assessed. RESULTS: Sixty and 180 min of breathing hyperoxic gas induced significant protection against severe ischemia and reperfusion-induced arrhythmias. Total number of premature ventricular beats was markedly attenuated by hyperoxia pre-exposure, especially in H60 and H180 groups. Duration of ventricular tachycardia and ventricular fibrillation was also affected by hyperoxia. Hyperoxia reduced the number of ventricular tachycardia episodes in ischemia and reperfusion phase. Accordingly, severity of arrhythmias (arrhythmia score) and infarct size were lower in hyperoxia-treated groups. The effects were more pronounced using hyperoxia immediately before harvesting the heart. CONCLUSION: These results indicate that hyperoxic preconditioning attenuates ventricular ischemia and reperfusion-induced arrhythmias in isolated rat hearts, decreases cardiac infarct size, and improves postischemic heart function. The effects seem to depend on the time course after hyperoxia treatment.

Normobaric hyperoxia induces ischemic tolerance and upregulation of glutamate transporters in the rat brain and serum TNF-alpha level.

Recent studies suggest that intermittent and prolonged normobaric hyperoxia (HO) results in ischemic tolerance to reduce ischemic brain injury. In this research, we attempted to see changes in excitatory amino acid transporters (EAATs) and TNF-alpha levels following prolonged and intermittent hyperoxia preconditioning. Rats were divided into four experimental groups, each of 21 animals. The first two were exposed to 95% inspired HO for 4 h/day for 6 consecutive days (intermittent HO, InHO) or for 24 continuous hours (prolonged HO, PrHO). The second two groups acted as controls, and were exposed to 21% oxygen in the same chamber. Each main group was subdivided to middle cerebral artery occlusion (MCAO-operated), sham-operated (without MCAO), and intact (without any surgery) subgroups. After 24 h from pretreatment, MCAO-operated subgroups were subjected to 60 min of right MCAO. After 24 h reperfusion, neurologic deficit score (NDS) and infarct volume were measured in MCAO-operated subgroups. EAATs expression and serum TNF-alpha levels were assessed in sham-operated and intact subgroups. Preconditioning with prolonged and intermittent HO decreased NDS and upregulated EAAT1, EAAT2, and EAAT3 and increased serum TNF-alpha levels significantly. Although further studies are needed to clarify the mechanisms of ischemic tolerance, the intermittent and prolonged HO seems to partly exert their effects via increase serum TNF-alpha levels and upregulation of EAATs.

Effects of chronic exercise on endothelial dysfunction and insulin signaling of cutaneous microvascular in streptozotocin-induced diabetic rats.

BACKGROUND: Abnormalities of the modulatory roles played by the endothelium and/or smooth muscle may be critical and initiating factors in the development of diabetic vascular disease. Decreased phosphatidylinositol 3-kinase (PI3-K)/Akt pathway activity and impaired nitric oxide production through this pathway may play pivotal roles in the diabetes-induced vascular dysfunction. Several findings have demonstrated that exercise training has therapeutic and protective effects in type 1 diabetes and could correct endothelial dysfunction. The molecular mechanisms, however, are only partially understood. METHOD: Male Wistar rats (220+/-10 g, N=60) were made diabetic by streptozotocin (60 mg/kg, subcutaneously). After 1 week of diabetes induction, animals were submitted to exercise training for 10 weeks on a treadmill. To characterize cutaneous microvascular responses by laser Doppler flowmetery, animals were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg) and placed on a heating pad. A rectal thermometer was inserted and body temperature was maintained at 37+/-0.5 degrees C. A tracheotomy was performed to minimize respiratory difficulties. Systemic arterial blood pressure and heart rate were measured by using a tail-cuff during assessment of cutaneous blood flow. RESULTS: (i) Acetylcholine-induced cutaneous perfusion were increased significantly by training in the diabetic groups; (ii) Cutaneous microvascular responses to sodium nitroprusside did not alter in control and diabetic animals by training; and (iii) Local microinjection of insulin increased cutaneous blood flow in trained diabetic and trained control rats compared with age-matched sedentary diabetic and sedentary control normal rats. The administration of wortmannin (PI3K inhibitor) and N-nitro-L-arginine ( nitric oxide synthase inhibitor) before insulin, however, attenuated the increase in cutaneous blood flow in trained diabetic and normal rats. CONCLUSIONS: Chronic exercise improved endothelium-dependent dilatation and potentiated insulin vascular function, possibly by PI3-kinase pathway in diabetic rats.