Diabetic nephropathy is a microvascular complication of diabetes. Its etiology involves metabolic disorder-induced endothelial dysfunction. Endothelium-derived nitric oxide (NO) plays an important role in a number of physiological processes, including glomerular filtration and endothelial protection. NO dysregulation is an important pathogenic basis of diabetic nephropathy. Hyperglycemia and dyslipidemia can lead to oxidative stress, chronic inflammation and insulin resistance, thus affecting NO homeostasis regulated by endothelial nitric oxide synthase (eNOS) and a conglomerate of related proteins and factors. The reaction of NO and superoxide (O₂^.-) to form peroxynitrite (ONOO^-) is the most important pathological NO pathway in diabetic nephropathy. ONOO^- is a hyper-reactive oxidant and nitrating agent in vivo which can cause the uncoupling of eNOS. The uncoupled eNOS does not produce NO but produces superoxide. Thus, eNOS uncoupling is a critical contributor of NO dysregulation. Understanding the regulatory mechanism of NO and the effects of various pathological conditions on it could reveal the pathophysiology of diabetic nephropathy, potential drug targets and mechanisms of action. We believe that increasing the stability and activity of eNOS dimers, promoting NO synthesis and increasing NO/ONOO^- ratio could guide the development of drugs to treat diabetic nephropathy. We will illustrate these actions with some clinically used drugs as examples in the present review.
Diabetes Mellitus ; Diabetic Nephropathies/drug therapy* ; Endothelium, Vascular ; Humans ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/therapeutic use* ; Oxidative Stress ; Peroxynitrous Acid/therapeutic use*

OBJECTIVE: To explore the therapeutic mechanism of tanshinone IIA in the treatment of pulmonary arterial hypertension (PAH) in rats. METHODS: A total of 100 male SD rats were randomized into 5 groups (n=20), and except for those in the control group with saline injection, all the rats were injected with monocrotaline (MCT) on the back of the neck to establish models of pulmonary hypertension. Two weeks after the injection, the rat models received intraperitoneal injections of tanshinone IIA (10 mg/kg), phosphatidylinositol 3 kinase (PI3K) inhibitor (1 mg/kg), both tanshinone IIA and PI3K inhibitor, or saline (model group) on a daily basis. After 2 weeks of treatment, HE staining and α-SMA immunofluorescence staining were used to evaluate the morphology of the pulmonary vessels of the rats. The phosphorylation levels of PI3K, protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS) in the lung tissue were determined with Western blotting; the levels of eNOS and NO were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of HE staining and α-SMA immunofluorescence staining showed that tanshinone IIA effectively inhibited MCT-induced pulmonary artery intimamedia thickening and muscularization of the pulmonary arterioles (P < 0.01). The results of Western blotting showed that treatment with tanshinone IIA significantly increased the phosphorylation levels of PI3K, Akt and eNOS proteins in the lung tissue of PAH rats; ELISA results showed that the levels of eNOS and NO were significantly decreased in the rat models after tanshinone IIA treatment (P < 0.01). CONCLUSION Treatment with tanshinone IIA can improve MCT-induced pulmonary hypertension in rats through the PI3K/Akt-eNOS signaling pathway.
Abietanes ; Animals ; Hypertension, Pulmonary/drug therapy* ; Male ; Monocrotaline/toxicity* ; Nitric Oxide Synthase Type III/therapeutic use* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg^-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg^-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg^-1 LIMK2i and 20.0 mg kg^-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.
Animals ; Apoptosis/drug effects* ; Arterial Pressure ; Electric Stimulation ; Erectile Dysfunction/pathology* ; Lim Kinases/antagonists & inhibitors* ; Male ; Nerve Crush ; Nitric Oxide Synthase Type III/metabolism* ; Penis/pathology* ; Peripheral Nerve Injuries/pathology* ; Phosphodiesterase 5 Inhibitors/therapeutic use* ; Phosphorylation ; Pyrimidines/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Sulfonamides/therapeutic use*

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

Erectile dysfunction (ED) associated with type 2 diabetes is a severe problem that requires effective treatment. Pancreatic kininogenase (PK) has the potential to improve the erectile function of ED patients. This study aims to investigate the effect of PK on erectile function in streptozotocin-induced type 2 diabetic ED rats. To achieve this goal, we divided male Sprague-Dawley rats into five groups. One group was not treated, and the other four groups were treated with saline, sildenafil, PK or sildenafil, and PK, respectively, for 4 weeks after the induction of type 2 diabetic ED. Then, intracavernous pressure under cavernous nerve stimulation was measured, and penile tissue was collected for further study. Endothelial nitric oxide synthase levels, smooth muscle content, endothelium content, cyclic guanosine monophosphate (cGMP) levels in the corpus cavernosum, and neuronal nitric oxide synthase levels in the dorsal penile nerve were measured. Improved erectile function and endothelium and smooth muscle content in the corpus cavernosum were observed in diabetic ED rats. When treating diabetic ED rats with PK and sildenafil at the same time, a better therapeutic effect was achieved. These data demonstrate that intraperitoneal injection of PK can improve erectile function in a rat model of type 2 diabetic ED. With further research on specific mechanisms of erectile function improvement, PK may become a novel treatment for diabetic ED.
Animals ; Cyclic GMP/metabolism* ; Diabetes Mellitus, Experimental/physiopathology* ; Erectile Dysfunction/physiopathology* ; Kallikreins/therapeutic use* ; Male ; Muscle, Smooth, Vascular/physiopathology* ; Nitric Oxide Synthase Type I/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Penile Erection/physiology* ; Penis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sildenafil Citrate/therapeutic use* ; Treatment Outcome ; Urological Agents/therapeutic use*

The study aimed to investigate the intervening role of Didang decoction (DDD) at different times in macrovascular endothelial defense function, focusing on its effects on the AMP-activated protein kinase (AMPK) signaling pathway. The effects of DDD on mitochondrial energy metabolism were also investigated in rat aortic endothelial cells (RAECs). Type 2 diabetes were induced in rats by streptozotocin (STZ) combined with high fat diet. Rats were randomly divided into non-intervention group, metformin group, simvastatin group, and early-, middle-, late-stage DDD groups. Normal rats were used as control. All the rats received 12 weeks of intervention or control treatment. Western blots were used to detect the expression of AMP-activated protein kinase α1 (AMPKα1) and peroxisome proliferator-activated receptor 1α (PGC-1α). Changes in the intracellular AMP and ATP levels were detected with ELISA. Real-time-PCR was used to detect the mRNA level of caspase-3, endothelial nitric oxide synthase (eNOS), and Bcl-2. Compared to the diabetic non-intervention group, a significant increase in the expression of AMPKα1 and PGC-1α were observed in the early-stage, middle-stage DDD groups and simvastatin group (P < 0.05). The levels of Bcl-2, eNOS, and ATP were significantly increased (P < 0.05), while the level of AMP and caspase-3 were decreased (P < 0.05) in the early-stage DDD group and simvastatin group. Early intervention with DDD enhances mitochondrial energy metabolism by regulating the AMPK signaling pathway and therefore may play a role in strengthening the defense function of large vascular endothelial cells and postpone the development of macrovascular diseases in diabetes.
AMP-Activated Protein Kinases ; metabolism ; Adenosine Triphosphate ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; Cardiovascular Diseases ; metabolism ; prevention & control ; Caspase 3 ; metabolism ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; complications ; drug therapy ; metabolism ; Diptera ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; Energy Metabolism ; drug effects ; Leeches ; Mitochondria ; drug effects ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Phytotherapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Prunus persica ; Rats, Sprague-Dawley ; Rheum ; Signal Transduction

OBJECTIVETo investigate the role of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway in erectile dysfunction (ED) caused by the absence of testosterone (T).

METHODSWe randomly divided 30 eight-week-old healthy male SD rats into groups A (control) , B (castration), and C (castration + androgen replacement). The rats in groups B and C were castrated surgically, and those in C injected with T undecanoate (100 mg/kg) at 1 week after castration, while the others with 0.9% normal saline instead. At 1 month after treatment, we determined the serum T level, intracavernous pressure (ICP), and mean carotid arterial pressure (MAP) of the rats, and detected the expressions of ERK1/2 and endothelial nitric oxide synthase (eNOS) by Western blot.

RESULTSThe serum T level was significantly lower in group B ([1.27 ± 0.48] nmol/L) than in A ([17.14 ± 1.07] nmol/L) and C ([16.24 ± 1.90] nmol/L) (P < 0.05), and so were ICP and MAP (P < 0.05). The expression of ERK1/2 showed no statistically significant differences among the three groups (P > 0.05), that of phosphatase ERK1/2 was markedly higher while that of eNOS remarkably lower in group B than in A and C (both P < 0.05).

CONCLUSIONAndrogen replacement may improve the erectile function of castrated rats by regulating the ERK1/2 pathway.

Androgens ; therapeutic use ; Animals ; Blotting, Western ; Erectile Dysfunction ; drug therapy ; metabolism ; Hormone Replacement Therapy ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Orchiectomy ; Penile Erection ; Penis ; Rats ; Rats, Sprague-Dawley ; Testosterone ; analogs & derivatives ; therapeutic use

BACKGROUNDDyslipidemia caused by liver injury is a significant risk factor for cardiovascular complications. Previous studies have shown that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO), and NO/endothelial nitric oxide synthase (eNOS) pathway is the key route of NO production. The purpose of this study was to investigate whether H2S can ameliorate the high blood pressure and plasma lipid profile in Nw-nitro-L-argininemethyl ester (L-NAME)-induced hypertensive rats by NO/eNOS pathway.

METHODSThirty-six 4-week old Sprague-Dawley (SD) male rats were randomly assigned to 6 groups (n = 6): control group, L-NAME group, control + glibenclamide group, control + NaHS group, L-NAME + NaHS group, and L-NAME + NaHS + glibenclamide group. Measurements were made of plasma triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (CHO), glutamic-pyruvic transaminase (ALT) levels after 5 weeks. Then measurements of NO level and proteins expression of eNOS, P-eNOS, AKT, P-AKT were made in liver tissue.

RESULTSAfter 5 weeks of L-NAME treatment, the blood pressure, plasma TG ((1.22±0.12) mmol/L in L-NAME group vs. (0.68±0.09) mmol/L in control group; P < 0.05) and LDL ((0.54±0.04) mmol/L in L-NAME group vs. (0.28±0.02) mmol/L in control group; P < 0.05) concentration were significantly increased, and the plasma HDL ((0.26±0.02) mmol/L in L-NAME group vs. (0.69±0.07) mmol/L in control group; P < 0.05) concentration significantly decreased. Meanwhile the rats treated with L-NAME exhibit dysfunctional eNOS, diminished NO levels ((1.36±0.09) mmol/g protein in L-NAME group vs. (2.34±0.06) mmol/g protein in control group; P < 0.05) and pathological changes of the liver. H2S therapy can markedly decrease the blood pressure ((37.25±4.46) mmHg at the fifth week; P < 0.05), and ameliorate the plasma TG ((0.59±0.06) mmHg), LDL ((0.32±0.04) mmHg), and HDL ((0.46±0.03) mmHg) concentration in L-NAME + NaHS group (all P < 0.05). H2S therapy can also restore eNOS function and NO bioavailability and attenuate the pathological changes in the liver in L-NAME-induced hypertensive rats.

CONCLUSIONH2S protects the L-NAME-induced hypertensive rats against liver injury via NO/ eNOS pathway, therefore decreases the cardiovascular risk.

Animals ; Cardiovascular Diseases ; metabolism ; prevention & control ; Hydrogen Sulfide ; therapeutic use ; Hypertension ; chemically induced ; drug therapy ; Liver ; drug effects ; metabolism ; Male ; NG-Nitroarginine Methyl Ester ; toxicity ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

This study investigated the role of angiotensin II receptor blocker in atrial remodeling in rats with atrial fibrillation (AF) induced by a myocardial infarction (MI). MIs were induced by a ligation of the left anterior descending coronary artery. Two days after, the rats in the losartan group were given losartan (10 mg/kg/day for 10 weeks). Ten weeks later, echocardiography and AF induction studies were conducted. Ejection fraction was significantly lower in the MI rats. Fibrosis analysis revealed much increased left atrial fibrosis in the MI group than sham (2.22 +/- 0.66% vs 0.25 +/- 0.08%, P = 0.001) and suppression in the losartan group (0.90 +/- 0.27%, P 0.001) compared with the MI group. AF inducibility was higher in the MI group than sham (39.4 +/- 43.0% vs 2.0 +/- 6.3%, P = 0.005) and significantly lower in losartan group (12.0 +/- 31.6%, P = 0.029) compared with the MI. The left atrial endothelial nitric oxide synthase (NOS) and sarco/endoplasmic reticulum Ca(2+)-ATPase levels were lower in the MI group and higher in the losartan group significantly. The atrial inducible NOS and sodium-calcium exchanger levels were higher in the MI and lower in the losartan group significantly. Losartan disrupts collagen fiber formation and prevents the alteration of the tissue eNOS and iNOS levels, which prevent subsequent AF induction.
Angiotensin Receptor Antagonists/*therapeutic use ; Animals ; Atrial Fibrillation/*prevention & control ; Atrial Remodeling ; Disease Models, Animal ; Fibrosis ; Heart Failure/*etiology/ultrasonography ; Immunohistochemistry ; Losartan/*therapeutic use ; Male ; Myocardial Infarction/*complications/ultrasonography ; Nitric Oxide Synthase Type II/metabolism ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Angiotensin/chemistry/metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism ; Sodium-Calcium Exchanger/metabolism

OBJECTIVETo study the protective mechanism of Huoxue Anxin Recipe (HAR) on peroxidation damage of acute myocardial infarction (AMI) rats.

METHODSThe AMI rat model was established by occluding the left anterior descending coronary artery. Compound Danshen Dripping Pill (CDDP) was used as the positive control. CDDP and HAR were administered to rats for 7 successive days since modeling. The heart function was detected using color Doppler echocardiography. Activities of induced nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), total superoxide dismutase (tSOD) activity, and contents of malondialdehyde (MDA) were detected by ultraviolet spectrophotometer method.

RESULTSCompared with the sham-operation group, ejection fraction (EF) and fraction shortening (FS) rate significantly decreased in the model group (P < 0.01). Compared with the model group, EF and FS rate significantly increased in the HAR group, showing statistical difference (P < 0.05). There was no statistical difference in activities of serum iNOS, eNOS, or tSOD among all groups (P > 0.05). Compared with the sham-operation group, iNOS activities and MDA contents significantly increased in the myocardial tissue of the model group (P < 0.01), activities of eNOS and tSOD significant decreased (P < 0.01). Compared with the model group, iNOS activities in the myocardial tissue, MDA contents both in serum and the myocardial tissue significantly decreased (P < 0.05), activities of eNOS and tSOD significantly increased in the HAR group (P < 0.05). There was no statistical difference in each index between the CDDP group and the HAR group (P > 0.05).

CONCLUSIONSHAR could significantly improve cardiac functions of AMI rats. Its roles might be associated with regulating imbalanced iNOS/eNOS expressions and alleviating peroxidation damage of the myocardial tissue.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Malondialdehyde ; metabolism ; Myocardial Infarction ; drug therapy ; metabolism ; Myocardium ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Oxidative Stress ; Phytotherapy ; Rats ; Rats, Wistar ; Superoxide Dismutase ; metabolism