The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 μg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.
Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Antiparkinson Agents ; pharmacology ; therapeutic use ; Cell Death ; drug effects ; Cell Line ; Disease Models, Animal ; Drugs, Chinese Herbal ; chemistry ; Neurons ; pathology ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type I ; biosynthesis ; Oxidopamine ; toxicity ; Paeonia ; chemistry ; Parkinsonian Disorders ; chemically induced ; drug therapy ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plants, Medicinal ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects ; enzymology ; Tyrosine 3-Monooxygenase ; genetics ; metabolism

OBJECTIVETo detect the expression of HO-2 in the corpus cavernosum of rats with chronic renal failure (CRF) , and investigate the role of HO-2 in penile erection and its association with testosterone.

METHODSFifteen 10-week-old SD rats underwent 5/6 kidney removal for the establishment of CRF models, and another 15 included as controls. Twelve weeks later, both the two groups of animals were subjected to electrostimulation of the cavernous nerve for the detection of intracavernosal pressure (ICP) and mean arterial pressure (MAP), and the protein contents of HO-2, nNOS and eNOS in the penile tissues were determined by Western blot and immunohistochemical analysis.

RESULTSThe ICPmax/MAP after 3 V and 5 V stimulation of the cavernous nerve was (0.121 +/- 0.084) and (0.135 +/- 0.088), the serum testosterone level was (1.190 +/- 0.946) nmol/L, and the expression of HO-2 was (0.510 +/- 0.397) in the CRF group, all significantly lower than in the control rats, which were (0.263 +/- 0.147 and 0.244 +/- 0.089), (7.800 +/- 5.001) nmol/L (P<0.01) and (2.672 +/- 1.720, P<0.01), respectively. There was a correlation between the decrease of HO-2 expression and the reduction of serum testosterone (r = 0.902, P < 0.01).

CONCLUSIONThe lowered level of serum testosterone and decreased contents of HO-2, eNOS and nNOS may play a role in CRF-induced ED.

Animals ; Blotting, Western ; Erectile Dysfunction ; complications ; physiopathology ; Heme Oxygenase (Decyclizing) ; biosynthesis ; Immunohistochemistry ; Kidney Failure, Chronic ; complications ; physiopathology ; Male ; Nitric Oxide Synthase Type I ; biosynthesis ; Nitric Oxide Synthase Type III ; biosynthesis ; Penis ; enzymology ; Rats ; Rats, Sprague-Dawley

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappa B activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappa B binding activity and p65 subunit levels in nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.
Animals ; Cell Death/drug effects ; Cell Line ; Cell Nucleus/metabolism ; Cell Survival/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/pharmacology ; I-kappa B Proteins/metabolism ; Insulin/secretion ; Insulin-Secreting Cells/*cytology/*drug effects/enzymology ; Interferon-gamma/*pharmacology ; Interleukin-1beta/*pharmacology ; Male ; NF-kappa B/*metabolism ; Nitric Oxide/biosynthesis ; Nitric Oxide Synthase Type II/genetics/metabolism ; Protein Transport/drug effects ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury.

METHODSMale SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting.

RESULTSnNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells.

CONCLUSIONLigustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Blotting, Western ; Brain ; blood supply ; drug effects ; enzymology ; Brain Ischemia ; complications ; Cell Proliferation ; drug effects ; Cerebral Ventricles ; blood supply ; drug effects ; pathology ; Dentate Gyrus ; blood supply ; drug effects ; pathology ; Immunohistochemistry ; Male ; Nerve Regeneration ; drug effects ; Nitric Oxide Synthase Type I ; biosynthesis ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; physiopathology

OBJECTIVE: To determine the effects of Tongxinluo on cell viability and tissue factor (TF) in AngII induced vascular endothelial cells and to investigate its mechanism. METHODS: AngII(10(-6)mol/L) was added to human vascular endothelial cells (HUVECs) culture media alone or with various concentration of Tongxinluo drug containing plasma (5%,10%, and 20%) added 30 minutes before AngII. Cell viability was evaluated after 24-hour incubation with AngII in a dose manner. TF, AngII type 1 receptor (AT(1)) mRNA, NO synthase (NOS) and NO were observed after 24-hour incubation with AngII. In addition, NOS inhibitor nomega-nitro-larginine (L-NAME) was added 30 minutes before Tongxinluo and AngII. Cell viability, TF, AT(1)mRNA, the level of NOS and NO were evaluated after 24-hour incubation with Tongxinluo and AngII. RESULTS: Tongxinluo significantly improved AngII induced endothelial cell viability and the effect was the most obvious at 10%. Tongxinluo (10%) decreased the TF and AT(1) mRNA while increased the NOS and NO levels. L-NAME obviously inhibited the effects of Tongxinluo on cell viability, TF, AT(1) mRNA, and NOS and NO levels. CONCLUSION Up-regulating NOS-NO signaling may be the mechanism of Tongxinluo on cell viability and TF in AngII induced vacular endothelial cells.
Angiotensin II ; pharmacology ; Cell Line ; Cell Survival ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Enzyme Inhibitors ; Enzyme-Linked Immunosorbent Assay ; Humans ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide Synthase Type I ; antagonists & inhibitors ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Angiotensin, Type 1 ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thromboplastin ; biosynthesis ; genetics

OBJECTIVE: To investigate the effect of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, on the activation of hepatic stellate cells (HSCs) and its mechanism. METHODS: Primary HSCs isolated from SD rats were cultured and treated with different concentrations (1, 3 or 10micromol/L) of ADMA for various periods (12 approximately 48h). Expression of alpha-smooth muscle actin (alpha-SMA) and synthesis of type-I collagens in HSC were determined. Messenger RNA levels of the transforming growth factor-beta1 (TGF-beta(1)) in the HSCs were determined using RT-PCR. Intracellular reactive oxidant species (ROS) production was measured using oxidant-sensitive fluorescent indicator. Activation of nuclear factor-kappaB (NF-kappaB) was detected by electrophoretic mobility shift assay (EMSA). RESULTS: ADMA could increase alpha-SMA-positive cells ratio and Type I collagens production of HSCs in a concentration- and time-dependent manner, concomitant with the increase of the TGF-beta(1) mRNA level. Treatment with ADMA (10micromol/L) significantly increased the intracellular ROS production and activated NF-kappaB. Such effects of ADMA on the level of TGF-beta(1) mRNA could be markedly attenuated by pretreatment with antioxidant pyrrolidine dithiocarbamate (25micromol/L). CONCLUSION ADMA can induce the HSC activation by increasing TGF-beta(1) expression through ROS-NF-kappaB-dependent pathway. Therefore, ADMA should be a novel and endogenous activator of HSC, which may be involved in the development of liver fibrosis.
Actins ; biosynthesis ; Animals ; Arginine ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Collagen Type I ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Hepatocytes ; cytology ; drug effects ; metabolism ; Male ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; genetics

OBJECTIVETo explore the impacts of denervation on the morphology and the expression of neuronal nitric oxide synthase (nNOS) of prostate of the adolescent rats.

METHODSAdolescent male SD rats were randomly divided into group A and group B. The right pelvic ganglion denervation was performed in group B with the help of surgical microscope, and group A received a sham operation. Five weeks later, the ventral prostates were obtained for morphologic observation, apoptosis detection and the evaluation of nNOS expression.

RESULTSA 30.8% reduction of right ventral prostate (RVP) fresh weight was found in group B. After denervation, histological features showed an overall decrease in the numbers of cells and cell height, and apoptosis indexes (AI) was significantly higher than that in group A (P <0.01), while the expression of nNOS decreased apparently (P < 0.01).

CONCLUSIONThe study indicates that denervation can cause apoptosis of the prostatic, and affect the prostate growth of the adolescent rat. During this process, nNOS plays an important role in the regulation of apoptosis.

Animals ; Apoptosis ; Denervation ; In Situ Nick-End Labeling ; Male ; Nitric Oxide Synthase Type I ; biosynthesis ; Prostate ; cytology ; innervation ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation

OBJECTIVE: To investigate the expression of neuronal nitric oxide synthase (nNOS) in spinal dorsal horn and the effect of intrathecal katemine on the expression of nNOS in the rats with formalin-induced pain. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into 4 groups (n=8 in each group): a control group (C), an intrathecal saline group (NS), a katemine 50 microg group (K1), and a katemine 100 microg group (K2). The rats that were anesthetized with 10% chlroral hydrate 300 - 350 mg/kg by abdominal injection were intrathecally inserted a microspinal catheter into the lumbar region according to the method of modified Yaksh. After 5 days ,the rats in Group NS, K1 and K2 were intrathecal 20 microL saline or 10 microL katemine (50, 100 microg) followed by 10 microL saline, respectively. Thirty minutes later, 5% formalin 50 microL was subcutaneously injected into the left hindpaw. Pain intensity scoring (PIS) was used to assess antinociceptive behavior within 1 h after the formalin injection. The expression of nNOS in the spinal dorsal horn of L5 segment was assayed using immunohistochemistry 24 h later. RESULTS: Compared with Group NS, PIS of Group K1 and K2 was decreased obviously (P<0.01) in the second phase of formalin pain. The number of immunoreactive soma and immunohistochemistic dying grade of nNOS in the spinal dorsal horn of L5 segment was higher in Group NS than that in Group C (P<0.01), but Group K1 and K2 were lower than Group NS (P<0.01). CONCLUSION There was significant antinociception of intrathecal katemine in rats with formalin pain. Intrathecal katemine significantly inhibited the increase of nNOS expression in the spinal dorsal horn of formalin-induced pain, suggesting nNOS plays an important role in nociceptive transmission and modulation of the spinal cord.
Analgesics ; administration & dosage ; Animals ; Formaldehyde ; Injections, Spinal ; Ketamine ; administration & dosage ; Male ; Nitric Oxide Synthase Type I ; biosynthesis ; genetics ; Pain ; chemically induced ; drug therapy ; metabolism ; Posterior Horn Cells ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The expression of nitric oxide synthase (NOS) isoforms in the ovaries of pigs was examined to study the involvement of nitric oxide, a product of NOS activity, in the function of the ovary. Western blot analysis detected three types of NOS in the ovary, including constitutive neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS); eNOS immunoreactivity was more intense compared with that of iNOS or nNOS. Immunohistochemical studies demonstrated the presence of nNOS and eNOS in the surface epithelium, stroma, oocytes, thecal cells, and endothelial cells of blood vessels. Positive immunoreactions for nNOS and iNOS were detected in the granulosa cells from multilaminar and antral follicles, but not in those of unilaminar follicles. iNOS was detected in the surface epithelium, oocytes, and theca of multilaminar and antral follicles. Taking all of the findings into consideration, the observed differential expression of the three NOS isoforms in the ovary suggests a role for nitric oxide in modulating reproduction in pigs.
Animals ; Blotting, Western/veterinary ; Female ; Immunohistochemistry/veterinary ; Nerve Tissue Proteins/*biosynthesis ; Nitric Oxide/metabolism ; Nitric Oxide Synthase/*biosynthesis ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Ovarian Follicle/*enzymology/growth&development ; Swine/*physiology

OBJECTIVETo investigate the effects of glycosides of tripterygium wilfordii (GTW), methyltestosterone and Zhuanggushenjin capsule on nitric oxide synthase (NOS) in rat testes.

METHODSForty-five rats were equally divided into 5 groups, and respectively given GTW [10 mg/(kg x d)], methyltestosterone [2 mg/(kg x d)], Zhuanggushenjin capsule [0.3 g/(kg x d)], distilled water plus Tween 80 (control I), and distilled water alone (control II) for 4 weeks. At the end of the 5th week, the immunochemical ABC method was used to observe the effects of the three drugs on the NOS positive Leydig cells of the rats.

RESULTSCompared with control II, the GTW group had a significant decrease in the numbers of nNOS and eNOS positive Leydig cells, the methyltestosterone group showed an increase in the number of nNOS but a decrease in that of eNOS positive Leydig cells, and the Zhuanggushenjin group had an increase in the numbers of both nNOS and eNOS positive Leydig cells.

CONCLUSIONGTW can reduce NO production by inhibiting eNOS and nNOS, and hence influence the spermatogenic process. Zhuanggushenjin capsule plays an important role in improving male sexual function by enhancing nNOS and eNOS expression and NO synthesis.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Leydig Cells ; drug effects ; enzymology ; Male ; Methyltestosterone ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type I ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis ; drug effects ; Tripterygium