Design, synthesis, and mechanistic insight of novel imidazolones as potential EGFR inhibitors and apoptosis inducers.

As regards to the structural analysis and optimization of diverse potential EGFR inhibitors, two series of imidazolyl-2-cyanoprop-2-enimidothioates and ethyl imidazolylthiomethylacrylates were designed and constructed as potential EGFR suppressors. The cytotoxic effect of the prepared derivatives was assessed toward hepatic, breast, and prostate cancerous cells (Hep-G2, MCF-7, and PC-3). Three derivatives 3d, 3e, and 3f presented potent antiproliferative activity and selectivity against the examined tumor cells showing IC50 values at low micromolar levels. Hence, successive biological assays were applied to determine the probable mechanism of action of the new compounds. They exhibited significant EGFR suppression with an IC50 range of 0.137-0.507 µM. The most effective EGFR inhibitor 3f arrested the MCF-7 cell cycle at the S phase by inducing the apoptotic pathway that was confirmed via increasing the expression of Caspases 8, 9, and Bax, which are associated with Bcl-2 decline. Additionally, molecular docking displayed a distinctive interaction between 3f and EGFR binding pocket. Overall, this work introduces some novel imidazolyl-2-cyanoprop-2-enimidothioates and ethyl imidazolylthiomethylacrylates as potential cytotoxic and EGFR inhibitors that deserve further research in tumor therapy.

Design and synthesis of novel dithiazole carboxylic acid Derivatives: In vivo and in silico investigation of their Anti-Inflammatory and analgesic effects.

Inflammation is a complex set of interactions that can occur in tissues as the body's defensive response to infections, trauma, allergens, or toxic compounds. Therefore, in almost all diseases, it can be observed because of primary or secondary reasons. Since it is important to control and even eliminate the symptoms of inflammation in the treatment of many diseases, anti-inflammatory and analgesic drugs are always needed in the clinic. Therefore, the discovery of new anti-inflammatory/analgesic drugs with increased effectiveness and safer side effect profiles is among the popular topics of medicinal chemistry. Therefore, in this study, in order to synthesize and diversify new molecules, we focused on the N,N-dithiazole carboxylic acid core and linked it with the chalcone functional group. The final eleven molecules were analyzed via HRMS, 1H NMR, and 13C NMR. The antinociceptive effects of the test compounds were examined by tail-clip, hot-plate, and formalin methods in mice, while their anti-inflammatory activities were investigated by carrageenan-induced inflammation tests in rats. The motor activities of the experimental animals were evaluated using an activity-meter device. Obtained findings revealed that none of the test compounds (10 mg/kg) were effective in the tail-clip and hot-plate tests. However, compounds 4b, 4c, 4f, 4 h, and 4 k in the serial shortened the paw-licking times of mice in the late phase of the formalin test indicating that these compounds had peripherally-mediated antinociceptive effects. The same compounds, moreover, showed potent anti-inflammatory effects by significantly reducing paw edema of rats in the inflammation tests. To provide an approach to pharmacological findings regarding possible mechanisms of action, the binding modes of the most active compounds were investigated by in silico approaches. The results of molecular docking studies indicated that the anti-inflammatory and analgesic activities of the compounds might be related to the inhibition of both COX-1 and COX-2 isoenzymes. Findings obtained from in silico studies showed that 4 k, which was chosen as a model for its analogs in the series, forms strong bindings to the basic residues (Arg120, Tyr355), side pocket loop area and deep hydrophobic regions of the enzyme. Moreover, results of the molecular dynamics simulation studies revealed that ligand-COX enzyme complexes are quite stable. Obtained results of in vivo and in silico studies are in harmony, and all together point out that compounds 4b, 4c, 4f, 4 h, and 4 k have significant anti-inflammatory and analgesic activities with good ADME profiles. The potential of the derivatives, whose pharmacological activities were revealed for the first time in this study, as anti-inflammatory and analgesic drug candidates, needs to be evaluated through comprehensive clinical studies.

Hyperbaric oxygen increases glioma cell sensitivity to antitumor treatment with a novel isothiourea derivative in vitro.

Glioblastoma (GBM) is the most common primary brain tumor. Tumor hypoxia is a pivotal factor responsible for the progression of this malignant glioma, and its resistance to radiation and chemotherapy. Thus, improved tumor tissue oxygenation may promote greater sensitivity to anticancer treatment. Protein kinase D1 (PKD1) protects cells from oxidative stress, and its abnormal activity serves an important role in multiple malignancies. The present study examined the effects of various oxygen conditions on the cytotoxic potential of the novel isothiourea derivate N,N'­dimethyl­S­(2,3,4,5,6­pentabromobenzyl)­â€‹isothiouronium bromide (ZKK­3) against the T98G GBM cell line. ZKK­3 was applied at concentrations of 10, 25 and 50 µM, and cells were maintained under conditions of normoxia, anoxia, hypoxia, hyperbaric oxygen (HBO), hypoxia/hypoxia and hypoxia/HBO. The proliferation and viability of neoplastic cells, and protein expression levels of hypoxia­inducible factor 1α (HIF­1α), PKD1, phosphorylated (p)PKD1 (Ser 916) and pPKD1 (Ser 744/748) kinases were evaluated. Oxygen deficiency, particularly regarding hypoxia, could diminish the cytotoxic effect of ZKK­3 at 25 and 50 µM and improve T98G cell survival compared with normoxia. HBO significantly reduced cell proliferation and increased T98G cell sensitivity to ZKK­3 when compared with normoxia. HIF­1α expression levels were increased under hypoxia compared with normoxia and decreased under HBO compared with hypoxia/hypoxia at 0, 10 and 50 µM ZKK­3, suggesting that HBO improved oxygenation of the cells. ZKK­3 exhibited inhibitory activity against pPKD1 (Ser 916) kinase; however, the examined oxygen conditions did not appear to significantly influence the expression of this phosphorylated form in cells treated with the tested compound. Regarding pPKD1 (Ser 744/748), a significant difference in expression was observed only for cells treated with 10 µM ZKK­3 and hypoxia/hypoxia compared with normoxia. However, there were significant differences in the expression levels of both phosphorylated forms of PKD1 under different oxygen conditions in the controls. In conclusion, the combination of isothiourea derivatives and hyperbaric oxygenation appears to be a promising therapeutic approach for malignant glioma treatment.

Inducible Nitric Oxide Synthase (iNOS) Mediates Vascular Endothelial Cell Apoptosis in Grass Carp Reovirus (GCRV)-Induced Hemorrhage.

Hemorrhage is one of the most obvious pathological phenomena in grass carp reovirus (GCRV) infection. The etiology of GCRV-induced hemorrhage is unclear. We found inducible nitric oxide synthase (iNOS) may relate to viral hemorrhage according to the previous studies, which is expressed at high levels after GCRV infection and is related to apoptosis. In this study, we aimed to investigate the mechanism of iNOS on apoptosis and hemorrhage at the cell level and individual level on subjects who were infected with GCRV and treated with S-methylisothiourea sulfate (SMT), an iNOS inhibitor. Cell structure, apoptosis rate, and hemorrhage were evaluated through fluorescence microscopy, Annexin V-FITC staining, and H&E staining, respectively. Cell samples and muscle tissues were collected for Western blotting, NO concentration measure, caspase activity assay, and qRT-PCR. iNOS-induced cell apoptosis and H&E staining showed that the vascular wall was broken after GCRV infection in vivo. When the function of iNOS was inhibited, NO content, apoptosis rate, caspase activity, and hemorrhage were reduced. Collectively, these results suggested iNOS plays a key role in apoptosis of vascular endothelial cells in GCRV-induced hemorrhage. This study is the first to elucidate the relationship between iNOS-induced cell apoptosis and GCRV-induced hemorrhage, which lays the foundation for further mechanistic research of virus-induced hemorrhage.

Preventive treatment with dizocilpine attenuates oedema in a carrageenan model of inflammation: the interaction of glutamatergic and nitrergic signaling.

Dizocilpine is a highly selective and potent non-competitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. It is well known that dizocilpine has different neuroprotective effects in animal models of pain, epilepsy and oedema during trauma. The search for alternative antiinflammatory drugs is ongoing. We investigated the anti-oedematous effects of dizocilpine and the probable mechanism of action in a rat model that mimics local and persistent inflammation without tissue injury or damage. Male Wistar rats were injected with 100 µL of 0.5% carrageenan to the plantar surface of the hind paw. Anti-oedematous activity was assessed in the carrageenan-induced paw inflammatory oedema test with a plethysmometer. To assess possible mechanisms of dizocilpine action, we examined the effects of the selective inhibitor of neuronal [N-ω-propyl-L-arginine hydrochloride (L-NPA)] and inducible [S-methylisothiourea (SMT)] nitric oxide synthase (NOS). Dizocilpine after systemic (0.0005, 0.005 and 0.02 mg/kg, subcutaneous (s.c.)), but not after local peripheral administration, reduced the paw inflammatory oedema. The effect is not dose dependent, and the highest decrease by about 47% at the time of maximally developed oedema was achieved with 0.005 mg/kg. Intraperitoneally (i.p.) administered L-NPA (0.5, 1 and 2 mg/kg) or SMT (0.005, 0.01 and 0.015 mg/kg) before dizocilpine abolished or reduced the anti-oedematous effect of dizocilpine by about 70-85%. An acute single dose of dizocilpine administered before inducing oedema systemically reduced the development of inflammatory oedema. The mechanism of the anti-oedematous effect includes, at least partially, an increase in nitric oxide (NO) production.

Involvement of inducible nitric oxide synthase (iNOS) in immune-functioning of Paphia malabarica (Chemnitz, 1782).

In recent years, the role of inducible nitric oxide synthase (iNOS) isoform has been widely studied because of its immunological relevance in higher organisms as well as invertebrates including bivalves. However, little is known about the immunological role of iNOS in Paphia malabarica defense mechanism. In this study, we immunodetected the presence of iNOS in P. malabarica hemocytes using antibody N9657 monoclonal anti-nitric oxide synthase. In addition, increased iNOS activity was evident in response to a higher bacterial dosage (Vibrio parahaemolyticus and V. cholerae), highlighting the dose-dependent iNOS activity induction. Also, higher bacterial survivability was observed in the presence of iNOS inhibitor, i.e., S-methylisothiourea hemisulphate (SMIS) thus, validating the bactericidal role of iNOS. These findings implicate the involvement of iNOS in immune-functioning of P. malabarica. Future work should focus on elucidating the expression and regulation of pathogenesis in P. malabarica, involving iNOS.

Potent Antitumour Effects of Novel Pentabromobenzylisothioureas Studied on Human Glial-derived Tumour Cell Lines.

BACKGROUND/AIM: Tumours of astroglial origin are the most common primary brain malignancy characterized by infiltrative growth and resistance to standard antitumour therapy. Glioma progression is thought to be related to various intracellular signal transduction pathways that involve the activation of protein kinases. Protein kinases play important roles in cell differentiation, proliferation, and survival. Recently, novel, specific inhibitors of constitutively active serine/threonine kinases and structurally similar isothiourea derivatives were suggested to induce apoptosis and inhibit proliferation in several types of human cancer cells. MATERIALS AND METHODS: In this study, we examined the cytotoxic and proapoptotic activities of selected modified pentabromobenzyl isothioureas (ZKKs) in an adult human glioblastoma (T98G) and a subependymal giant cell astrocytoma cell (SEGA) line. We evaluated cell proliferation, viability, and apoptosis. RESULTS: Two pentabromobenzyl isothiourea bromide derivatives, ZKK-13 and N,N,N'-trimethyl-ZKK1 (TRIM), exhibited the most potent cytotoxic and proapoptotic efficacies against human glioma-derived cells, even at a very low concentration (1 µM). ZKK-13 (25-50 µM) inhibited cell growth by approximately 80-90% in 24 and 48 h of treatment. We showed that selected ZKKs exerted antiproliferative activity against astroglial neoplastic cells of both low- and high-grade tumour malignancy classes. No synergistic effects were detected when ZKKs were combined with serine/threonine kinase inhibitors. CONCLUSION: Our findings indicated that modified ZKKs show promise for the treatment of glioma-derived brain tumours.

A new promising simultaneous approach for attenuating type II diabetes mellitus induced neuropathic pain in rats: iNOS inhibition and neuroregeneration.

In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance nerve regeneration as well as prevent nerve degeneration. So, in the present study, we have investigated the effect of S-Methylisothiourea Sulfate (selective iNOS inhibitor) and Citicoline, alone and in combination, on Type II diabetes mellitus induced neuropathic pain in wistar rats. Type II diabetes was induced by providing high fat diet and low dose of Streptozotocin for 35 days in rats. Type II diabetes mellitus was assessed in terms of increased glucose, triglycerides, cholesterol, LDL levels, glucose tolerance and decrease in HDL levels. Neuropathy as the complication of type II diabetes was assessed in terms of decreased nerve conduction velocity, mechanical and thermal hyperalgesia and cold allodynia. Oxidative stress was assessed in sciatic nerve and showed increase in LPO and nitrite levels whereas decrease was shown in GSH and catalase activity. Axonal degeneration marked by nerve fibre dearrangement and demyelination was observed in histopathological studies. SMT (iNOS inhibitor), Citicoline and low dose combination of both drugs significantly attenuates the diabetic neuropathic pain assessed in terms of parameters employed. Thus, it may be concluded that simultaneous administration of SMT and Citicoline may provide potential therapeutics for diabetic neuropathic pain.

Cardiovascular risk and the effect of nitric oxide synthase inhibition in female rats: The role of estrogen.

It is known that autonomic modulation is responsive to ovarian hormone levels and that estrogen increases nitric oxide (NO) bioavailability. However, little is known about the interaction of nitric oxide synthase (NOS) isoforms with autonomic modulation, oxidative stress and cardiovascular risk in females. This study aimed to investigate cardiovascular, autonomic and oxidative parameters after selective NOS inhibition. A spectral analysis of systolic arterial pressure (SAP) and heart rate variability (HRV) was performed. NO levels, total antioxidant capacity (TRAP), lipid hydroperoxides (LOOH) and paraoxonase 1 (PON1) activity were measured in the plasma of rats treated with L-NG-nitroarginine methyl ester (L-NAME), S-methylisothiourea (SMT) or saline. Wistar rats, ovariectomized (OVX) with or without estradiol treatment (1mg/kg/day) or with a false ovariectomy (SHAM), were submitted to artery and vein catheterization. Cardiovascular parameters were evaluated before and after the administration of saline or NOS inhibitors. After 2h, plasma samples were collected for biochemical measurement. At baseline, cardiovascular and autonomic parameters were not different among the groups. L-NAME, the constitutive NOS isoform (cNOS) inhibitor, promoted an increase in mean arterial pressure (MAP) and a reduction in the low frequency band (LF) of SAP of SHAM rats, but this increase was smaller in OVX animals, which also showed a reduction in PON1 activity. The decreased activity of PON1 caused by L-NAME was prevented in the OVX+E group. SMT, an inducible NOS isoform (iNOS) inhibitor, promoted an increase in MAP and in the LF of SAP, in interbeat interval (IBI) parameters at LFnu and in LF/HF ratio of HRV in all groups, but the OVX+E had lower levels of NO when compared with the OVX group. Our data suggest that while cNOS contributes to maintaining the activity of PON1 in OVX rats, iNOS activity maintains the levels of NO in OVX+E rats.

Inhibition of neuronal and inducible nitric oxide synthase does not affect the analgesic effects of NMDA antagonists in visceral inflammatory pain.

Previously we described the antinociceptive effect of magnesium sulfate and dizocilpine (MK-801) in the visceral and somatic rat models of pain. In the somatic model of pain, we established the influence of selective inhibitors of neuronal and inducible nitric oxide synthase on the antihyperalgesic effects of magnesium sulfate and dizocilpine. Therefore, the objective of the present study was to determine in the rat model of visceral pain whether same mechanisms are involved in the antinociceptive action of magnesium sulfate and dizocilpine. Analgesic activity was assessed using the acetic acid-induced writhing test in rats. Subcutaneous injection of either magnesium sulfate (15 mg/kg) or dizocilpine (0.01 mg/kg) decreased the number of writhes by about 60 and 70%, respectively. The role of nitric oxide on the effects of magnesium sulfate and dizocilpine was evaluated using selective inhibitor of neuronal (N-ω-Propyl-L-arginine hydrochloride (L-NPA)) and inducible (S-methylisothiourea (SMT)) nitric oxide synthase, which per se did not affect the number of writhes. We observed that the antinociceptive effect of magnesium sulfate or dizocilpine did not change in the presence of L-NPA (2 and 10 mg/kg, i.p.) and SMT (0.015 and 10 mg/kg, i.p.). We conclude that, nitric oxide produced by neuronal and inducible nitric oxide synthase does not modulate the effects of magnesium sulfate and dizocilpine in the visceral inflammatory model of pain in the rat.

Endogenous nitric oxide derived from NOS I or II in thoracic spinal cord exerts opposing tonic modulation on sympathetic vasomotor tone via disparate mechanisms in anesthetized rats.

The sympathetic preganglionic neurons (SPN) in the thoracic spinal cord regulate vasomotor tone via norepinephrine released from sympathetic terminals and adrenal medulla. We assessed the hypothesis that nitric oxide synthase I (NOS I)- and NOS II-derived nitric oxide (NO) in the thoracic spinal cord differentially modulate sympathetic outflow and that the adrenal medulla may be involved in those modulatory actions. In Sprague-Dawley rats, NOS I immunoreactivity was distributed primarily in the perikaryon, proximal dendrites, or axons of SPN, and small clusters of NOS II immunoreactivity impinged mainly on the circumference of SPN. Intrathecal administration of 7-nitroindazole (7-NI), a specific NOS I antagonist, into the thoracic spinal cord significantly reduced arterial pressure, heart rate, and basal or baroreflex-mediated sympathetic vasomotor tone. On the other hand, intrathecal application of S-methylisothiourea (SMT), a specific NOS II antagonist, elevated arterial pressure with a transient reduction of heart rate, induced a surge of plasma norepinephrine, and reduced baroreflex-mediated but not basal sympathetic vasomotor tone. Bilateral adrenalectomy significantly exacerbated the cardiovascular responses to 7-NI but antagonized those to SMT. We conclude that both NOS I and NOS II are present in the thoracic spinal cord and are tonically active under physiological conditions. Furthermore, the endogenous NO generated by NOS I-containing SPN exerts a tonic excitatory action on vasomotor tone mediated by norepinephrine released from the adrenal medulla and sympathetic nerve terminals. On the other hand, NO derived from NOS II exerts a tonic inhibitory action on sympathetic outflow from the SPN that targets primarily the blood vessels.

Nitric Oxide Produced by Macrophages Inhibits Adipocyte Differentiation and Promotes Profibrogenic Responses in Preadipocytes to Induce Adipose Tissue Fibrosis.

Fibrosis of adipose tissue induces ectopic fat accumulation and insulin resistance by inhibiting adipose tissue expandability. Mechanisms responsible for the induction of adipose tissue fibrosis may provide therapeutic targets but are poorly understood. In this study, high-fat diet (HFD)-fed wild-type (WT) and iNOS(-/-) mice were used to examine the relationship between nitric oxide (NO) produced by macrophages and adipose tissue fibrosis. In contrast to WT mice, iNOS(-/-) mice fed an HFD were protected from infiltration of proinflammatory macrophages and adipose tissue fibrosis. Hypoxia-inducible factor 1α (HIF-1α) protein level was increased in adipose tissue of HFD-fed WT mice, but not iNOS(-/-) mice. In contrast, the expression of mitochondrial biogenesis factors was decreased in HFD-fed WT mice, but not iNOS(-/-) mice. In studies with cultured cells, macrophage-derived NO decreased the expression of mitochondrial biogenesis factors, and increased HIF-1α protein level, DNA damage, and phosphorylated p53 in preadipocytes. By activating p53 signaling, NO suppressed peroxisome proliferator-activated receptor γ coactivator 1α expression, which induced mitochondrial dysfunction and inhibited preadipocyte differentiation in adipocytes. The effects of NO were blocked by rosiglitazone. The findings suggest that NO produced by macrophages induces mitochondrial dysfunction in preadipocytes by activating p53 signaling, which in turn increases HIF-1α protein level and promotes a profibrogenic response in preadipocytes that results in adipose tissue fibrosis.

The Effect of the iNOS Inhibitor S-Methylisothiourea and Hyperbaric Oxygen Treatment on Radiation Colitis in Rats.

INTRODUCTION: External radiotherapy is one of the main treatment modalities for a variety of malignancies. However, the lower gastrointestinal tract is sensitive to the ionizing radiation. Hyperbaric oxygen treatment (HOT) has been suggested as a viable treatment for refractory radiation colitis, but the effect of S-Methylisothiourea (SMT) in the radiation colitis have not reported. To investigate the effect of SMT, HOT and the combination of both in an acute radiation-induced enterocolitis model. METHODS: Sixty Sprague-Dawley rats were divided randomly into five equal groups. A single dose of gamma irradiation (25 Gy) was administered through the colorectal region to anesthetized rats. In the control group, we applied 2 ml of saline solution intraperitoneally for five days. In the HOT group, 100-per-cent oxygen at 2.5 atm pressure was applied for five days. In the SMT group, 10 mg/kg/day of SMT was applied intraperitoneally for five days. In the HOT+SMT group, HOT and SMT were both applied in the same dosages as in the preceding two groups. At the end of five days, the rats were sacrificed and colon samples were collected for histological grading. Blood samples were collected to test for : tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), IL-1ß, transforming growth factor-ß (TGF-ß) and intercellular adhesion molecule-1 (ICAM-1) mRNA. RESULTS: The TNF-α, IL-1ß, IL-10 and TGF-ß levels were reduced by SMT, HOT and HOT+SMT applications (p < 0.05). However ICAM-1 mRNA levels were not significantly lower (p:0.19). The microscopic scores differed significantly between the SMT, HOT and HOT+SMT groups and the control group. There was significant improvement histologically, especially in the HOT+SMT group. When we compared the weight of the rats before and after the study, weight loss was significantly lower in the SMT, HOT and HOT+SMT groups compared with the control group (p < 0.05). CONCLUSION: HOT and SMT together were significantly more effective in preventing weight loss and in reducing inflammation and the severity of colitis histology when compared with HOT and SMT separately.

Cochliomycin A inhibits the larval settlement of Amphibalanus amphitrite by activating the NO/cGMP pathway.

Cochliomycin A is a compound with anti-barnacle settlement activity and low toxicity, but the molecular mechanism of the compound is unknown. Here, isobaric tags for the relative or absolute quantitation (iTRAQ) labeling proteomic method were applied to analyze changes in the proteome of Amphibalanus (=Balanus) amphitrite cyprids in response to cochliomycin A treatment. Cochliomycin A affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways, among which the NO/cGMP pathway was considered to play a key role in barnacle larval settlement, while the cytochrome P450 and the GST pathways are mainly for detoxification. The results of real-time PCR further suggested the NO/cGMP pathway was activated in response to cochliomycin A. Larval settlement assays revealed that S-methylisothiourea sulfate (SMIS) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) rescued cyprids from cochliomycin A-induced inhibition of larval settlement. The findings supported the hypothesis that cochliomycin A inhibited barnacle larval settlement by stimulating the NO/cGMP pathway.

[A MODULATORY INFLUENCE OF THE iNOS ON THE FUNCTIONAL ACTIVITY OF THE KATP-CHANNELS IN CORONARY VESSELS OF RATS ADAPTED TO THE STRESS].

In the experiments carried out on the isolated by Langendorff's method hearts which were perfused at the constant coronary flow it was studied influence of iNOS-derived NO on the functional activity of the KATP-channels in the coronary vascular smooth muscle cells following formation of the adaptation to the stress. As an adaptation to the short stress as a 6-hours immobilization stress result in increase of NO production and rise of expression of gene iNOS; however, adaptation in contrast to the stress leads to moderate accumulation of iNOS against the background unchanged systemic activity of еNOS and in the absence of systemic low-grade inflammation. After an adaptation to the short stress or after 6 hours of immobilization the functional glybenclamid-inhibiting activity of the KATP-channels was suppressed. Nevertheless, after prolonged immobilization suppressed activity of the channels was abolished after blockage of iNOS, whereas iNOS blockage in adapted animals leads not only to the recovery but even to the hyperactivation of the KATP-channels. Hence, increase in NO production which is typical for the adaptation, limits activation of the KATP-channels thus creating a strong link between these channels activity and NO produced by iNOS.

Effects and Mechanism of Action of Inducible Nitric Oxide Synthase on Apoptosis in a Rat Model of Cerebral Ischemia-Reperfusion Injury.

Inducible nitric oxide synthase (iNOS) is a key enzyme in regulating nitric oxide (NO) synthesis under stress, and NO has varying ability to regulate apoptosis. The aim of this study was to investigate the effects and possible mechanism of action of iNOS on neuronal apoptosis in a rat model of cerebral focal ischemia and reperfusion injury in rats treated with S-methylisothiourea sulfate (SMT), a high-selective inhibitor of iNOS. Seventy-two male Sprague-Dawley (SD) rats were randomly divided into three groups: the sham, middle cerebral artery occlusion (MCAO) + vehicle, and MCAO + SMT groups. Neurobehavioral deficits, infarct zone size, and cortical neuron morphology were evaluated through the modified Garcia scores, 2,3,5-triphenyltetrazolium chloride (TTC), and Nissl staining, respectively. Brain tissues and serum samples were collected at 72 hr post-reperfusion for immunohistochemical analysis, Western blotting, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labeling assay (TUNEL) staining, and enzyme assays. The study found that inhibition of iNOS significantly attenuated the severity of the pathological changes observed as a result of ischemia-reperfusion injury: SMT reduced NO content as well as total nitric oxide synthase (tNOS) and iNOS activities in both ischemic cerebral hemisphere and serum, improved neurobehavioral scores, reduced mortality, reduced the infarct volume ratio, attenuated morphological changes in cortical neurons, decreased the rate of apoptosis (TUNEL and caspase-3-positive), and increased phospho (p)-AKT expression in ischemic penumbra. These results suggested that inhibition of iNOS might reduce the severity of ischemia-reperfusion injury by inhibiting neuronal apoptosis via maintaining p-AKT activity.

[INVOLVEMENT OF NO-SYNTHASE IN THE INFARCT REDUCING EFFECT OF CONTINUOUS CHRONIC NORMOBARTC HYPOXTA].

It was investigated the role of inducible and endothelial NO-synthase (NOS) in the infaret reducing effect of chronic continuous normobaric hypoxia (CCNH) on the model of coronary artery occlusion and reperfusion in rats. Rats were subjected to hypoxic exposure (12% O2 during 21 days). It has found that CCNH causes an increase in total levels of nitrate and nitrite in deproteinized blood serum in 1.5-fold and 2-fold in myocardium compared with intact animals. This effect is manifested in intact animals and in rats with a 20 minute coronary artery occlusion and reperfusi- on. Chronic continuous normobaric hvoxia exhibited infarct soaring effect. which does not manifest after pretreatment with NO-synthase inhibitor NAME (10 mg/kg intravenously), the selective inhibitor of inducible NOS S-methylisothiourea (3 mg/kg intraperitoneally), but remained after blocking neuronal NOS with 7-nitronidazol (50 mg/kg intravenously). The findings suggest that the inducible NO-synthase and nitric oxide play an important role in the implementation of the infaret limiting effect of chronic continuous normobaric hvnoxia.

Attenuation of bleomycin induced lung fibrosis by erdosteine and inhibition of the inducible nitric oxide synthase.

BACKGROUND AND OBJECTIVES: Despite advances in treatment modalities, the discovery of optimal medical therapies still remains a necessity in the management of pulmonary fibrosis. MATERIAL AND METHODS: The experiments were performed in 35 adult Sprague Dawley rats, randomly allotted into one of five groups (n=7). The control group was treated with 1 ml/kg, 0.9 % saline; the BLM group was given a single dose of BLM (2.5 U/kg); the BLM+ER group was treated with ER (10 mg/kg/day po) for 14 days after BLM administration; the BLM+SMT group was treated with i.p injections of SMT (20 mg/kg/ day) for 14 days after BLM administration; the BLM+ER+SMT group was treated with ER and SMT for 14 days after BLM administration. At the end of day 14, the results of histopathological, biochemical, and immunohistochemical investigations were analyzed. RESULTS: Serum TNF-α, nitrate/nitrite, and TBARS levels significantly increased in BLM group compared to control group (p < 0.001, p < 0.001 and p < 0.05 respectively). Lung tissue content of IL-6 was found to be lower in BLM+ER, BLM+SMT and BLM+ER+SMT groups compared to BLM group by immunhistochemical examinations (p < 0.01, p < 0.01 and p < 0.001, respectively). Similarly, the TNF-α reactions (p < 0.01 for each group) and NF-kB expressions were shown to be significantly different among the study groups (p < 0.05, p < 0.05 and p < 0.001, respectively). CONCLUSION: Based on our study, ER and SMT attenuate BLM-induced pulmonary fibrosis; the combination of two agents has a greater protective efficacy against fibrosis than one alone, reducing the inflammatory markers (Tab. 2, Fig. 2, Ref. 31).

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription.

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor N(É·)-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50µM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10µM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5µM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10µM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

The anti-apoptotic effect of polypeptide from Chlamys farreri (PCF) in UVB-exposed HaCaT cells involves inhibition of iNOS and TGF-ß1.

To investigate the molecular mechanisms of polypeptide from Chlamys farreri (PCF)'s anti-apoptotic effect, HaCaT cells were exposed to 20 mJ/CM(2) UVB, with or without pretreatment of TGF-ß1 antagonist SB431542, inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate (SMT), nitric oxide scavenger carboxy-PTIO, or 1.42, 2.84, and 5.69 mM PCF, or iNOS transfection (without UVB exposure). Apoptosis was confirmed with Hoechst 33258 staining; RT-PCR and western blot were used to determine the expression levels of iNOS and TGF-ß1 signaling pathway. Both UVB exposure and iNOS transfection-induced apoptosis in UVB-exposed HaCat cells, while PCF, SB431542, SMT, and carboxy-PTIO all inhibited UVB-induced apoptosis. TGF-ß1, Smad4, and Smad7 mRNA levels were all altered, similarly, iNOS, TGF-ß1, and pSmad2/3 protein levels were all altered in UVB-exposed HaCaT cells. In pretreated cells, SB431542, SMT, carboxy-PTIO, and 1.42-5.69 mM PCF all inhibited UVB-induced apoptosis. Moreover, PCF treatment inhibited the expression levels of iNOS, TGF-ß1, pSmad2/3, and Smad4, while increased the expression level of Smad7. SB431542 did not significantly alter iNOS expression, while SMT and carboxy-PTIO significantly altered TGF-ß1 signaling level. The anti-apoptotic effect of PCF in UVB-exposed HaCaT cells involves the inhibition of iNOS expression and subsequently inhibition of TGF-ß1 signaling pathway.