Role of micronucleus-activated cGAS-STING signaling in antitumor immunity / 浙江大学学报·医学版

cGAS-STING signaling is a significant component of the innate immune system and functions as a vital sentinel mechanism to monitor cellular and tissue aberrations in microbial invasion and organ injury. cGAS, a cytosolic DNA sensor, is specialized in recognizing abnormally localized cytoplasmic double-stranded DNA (dsDNA) and catalytically synthesizes the second messenger cyclic-GMP-AMP (cGAMP), which initiates a cascade of type I interferon and inflammatory responses mediated by STING. Micronucleus, a byproduct of chromosomal missegregation during anaphase, are also significant contributors to cytoplasmic dsDNA. These unstable subcellular structures are susceptible to irreversible nuclear envelope rupture, exposing genomic dsDNA to the cytoplasm, which potently recruits cGAS and activates STING-mediated innate immune signaling and its downstream activities, including type I interferon and classical nuclear factor-κB (NF-κB) signaling pathways lead to senescence, apoptosis, autophagy activating anti-cancer immunity or directly killing tumor cells. However, sustained STING activation-induced endoplasmic reticulum stress, activated chronic type I interferon and nonclassical NF-κB signaling pathways remodel immunosuppressive tumor microenvironment, leading to immune evasion and facilitating tumor metastasis. Therefore, activated cGAS-STING signaling plays a dual role of suppressing or facilitating tumor growth in tumorigenesis and therapy. This review elaborates on research advances in mechanisms of micronucleus inducing activation of cGAS-STING signaling and its implications in tumorigenesis and therapeutic strategies of malignant tumors.

Progress in DNA-sensing pathways in Epstein-Barr virus infection / 中华微生物学和免疫学杂志

Epstein-Barr virus (EBV) is generally susceptible in human beings and multi-organ systems can be involved in EBV infection, such as blood, respiratory, urinary, digestive and nervous systems. EBV infection also plays an important role in the pathogenesis of related tumors, autoimmune diseases and other diseases, posing a great threat to human health. As a DNA virus, EBV can be sensed by DNA recognition receptors to trigger a series of downstream immune responses. A DNA-sensing pathway consists of DNA sensors, adaptor molecules and downstream effector signals. Double-stranded DNA sensors mainly include absent in melanoma 2-like receptors (ALRs) and cyclic GMP-AMP synthase (cGAS). Adaptors were mainly stimulator of interferon genes (STING) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Downstream immune responses mainly involve typeⅠIFN, inflammasomes and proinflammatory cytokines. As a double-stranded DNA virus of the Herpesviridae family, EBV triggers complex innate and adaptive immune responses in the host, especially the sensing pathways mediated by a variety of DNA recognition receptors, which play a key role in host immune defense and pathogen immune evasion. This review made the DNA sensor as the clue to comprehensively summarize the progress in the activation, regulatory mechanism and clinical relevance of DNA-sensing pathways in EBV infection in recent years, aiming to achieve a better understanding of the host innate immune responses during EBV infection and provide an immunological basis for the prevention and treatment of EBV infection-related diseases.

Advances in cGAS-STING pathway and its inhibitors in immune and inflammatory diseases / 中国药理学通报

The cGAS-STING signaling pathway is one of the main pathways of immune defense against many types of pathogens. cGAS catalyzes the production of the second messenger cGAMP (cyclic GMP-tVMP) by recognizing plasma DNA and cGAMP subsequently binds to the interferon gene stimulating factor (STING). The pathway induces the production of type I interferon (IFN-I) and activates the innate immune system. The activation of the cGAS-STI]NG pathway could facilitate self-protection,thus STI]NG agonists for tumor immunotherapy have attracted much attention in recent years,and several drug candidates have been in clinical trials. Meanwhile,aberrant activation of cGAS-STI]NG could lead to autoimmune diseases and has attracted extensive interest in developing its inhibitors. This paper summarizes the mechanism and regulatory sites of the cGAS-STI]NG pathway,and outlines the research progress of cGAS-STING pathway-related immune and inflammatory diseases and its inhibitors.

Effect of HBsAg on the production of interferon-α in peripheral blood plasmacytoid dendritic cells induced by the stimulator of interferon genes signaling pathway / 临床肝胆病杂志

ObjectiveTo investigate the effect of HBsAg on the expression of interferon-α (IFN-α) in peripheral blood plasmacytoid dendritic cells (pDCs) induced by the stimulator of interferon genes (STING) signaling pathway activated by cyclic GMP-AMP (cGAMP). MethodPeripheral venous blood was collected from healthy adults and the patients with chronic hepatitis B virus (HBV) infection who attended the outpatient service or were hospitalized in Department of Infectious Diseases, The Affiliated Hospital of Xuzhou Medical University, from February to December 2016, and peripheral blood mononuclear cells (PBMCs) were isolated and extracted. After the STING agonist cGAMP was added to PBMCs, ELISA was used to measure the levels of IFN-α, interferon-β, and tumor necrosis factor-α in supernatant. PBMCs from healthy adults were pre-incubated with HBsAg and then stimulated by cGAMP, and supernatant was collected to measure IFN-α. The magnetic-activated cell sorting method was used to remove pDCs from PBMCs, and after culture with cGAMP, ELISA was used to measure the level of IFN-α in supernatant. PBMCs from healthy adults were stimulated by HBsAg and/or cGAMP, and then flow cytometry was used to measure the frequency of pDCs. The independent samples t-test was used for comparison of continuous data between two groups. ResultsPBMCs from the patients with chronic HBV infection stimulated by cGAMP in vitro had a significantly lower level of IFN-α than healthy controls (469.72±18.95 vs 599.90±84.06, t=4.868, P=0.001). PBMCs from healthy adults co-cultured with HBsAg and stimulated by cGAMP had a significantly lower level of IFN-α than those in the non-HBsAg group (448.5±52.0 vs 571.0±30.8, t=4.500, P=0.011). Compared with PBMCs containing pDCs, PBMCs without pDCs stimulated by cGAMP had a significant reduction in the level of IFN-α (164.50±40.73 vs 339.50±35.33, t=6.482, P=0.001). Compared with PBMCs from healthy adults stimulated by cGAMP, PBMCs pre-incubated with HBsAg and then stimulated by cGAMP had a significant reduction in the frequency of pDCs (0.12%±0.04% vs 0.24%±0.04%, t=5.176, P=0.014). ConclusionHBsAg can inhibit the expression of IFN-α induced by the STING pathway in pDCs activated by cGAMP.

cGAS recognizes HTLV-1 RTI and induces STING-dependent innate immune responses / 中华微生物学和免疫学杂志

Objective To investigate whether cyclic GMP-AMP synthase ( cGAS ) , a cytosolic DNA sensor, could recognize the reverse transcription intermediate and induce the subsequent signaling path-way during the infection of human T cell leukemia virus type 1 ( HTLV-1 ) . Methods Biotin-labeled ssDNA90, a reverse transcription intermediate of HTLV-1, was transfected into HeLa cells and the interac-tion between it and cGAS was detected by co-immunoprecipitation experiments. HeLa cells were co-cultured with HTLV-1-positive MT2 cells and the interaction between cGAS and stimulator of interferon genes ( STING) was analyzed by co-immunoprecipitation experiments. The expression of STING in HeLa cells was silenced by siRNA. cGAS was transfected into the HeLa cells 24 h after the silencing and after 24 h, these cells were co-cultured with MT2 cells for another 24 h. Real-time PCR assay was used to measure the ex-pression of IFN-β, RANTES ( regulated upon activation, normal T-cell expressed, and secreted) , TNF-α, HTLV-1 protein Tax, p19 and HBZ. Immunoblot assay was performed to evaluate the phosphorylation of IRF3 and p65 in HeLa cells. Results cGAS interacted with ssDNA90. cGAS interacted with STING in the cytoplasm. In STING-silenced HeLa cells, cGAS transfection had no influence on the expression of IFN-β, RANTES , TNF-α, Tax , p19 or HBZ , nor did it affect the phosphorylation of IRF3 or p65 . Conclusions cGAS interacted with HTLV-1 RTI ssDNA90 and activated STING-dependent innate immune responses.

Research progress of cGAS-STING signaling pathway in anti-DNA virus immunity / 中国比较医学杂志

Innate immune system rapidly detects and responds to viruses at the early stage of viral infection. However,the mechanisms by which the immune system recognizes and eliminates them have not been fully clarified so far. Studies have shown that receptors are the primary tool for cell recognition and detection of viruses, and cyclic GMP-AMP synthase(cGAS)is one of the newly found DNA recognition receptors. cGAS transmits the signal to the downstream protein called STING(stimulator of interferon genes)and mediates the production of type I interferon(IFN-I),thereby to initiates the antiviral immunity of cells. This review briefly introduces the mechanism of the cGAS-STING signaling pathway, in order to provide a theoretical basis for the research and development of new antiviral drugs.

Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues.</p><p><b>METHODS</b>The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry.</p><p><b>RESULTS</b>P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues.</p><p><b>CONCLUSIONS</b>Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
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Cyclic GMP-AMP synthase ( cGAS) inhibits the replication of human T cell leukemia virus type 1 (HTLV-1) in HeLa cells through regulating innate immune response / 中华微生物学和免疫学杂志

Objective To investigate the function and the possible mechanism of cyclic GMP-AMP synthase (cGAS), a DNA sensor, in HeLa cells during human T cell leukemia virus type 1 (HTLV-1) in-fection.Methods HeLa cells were co-cultured with MT2 cells (HTLV-1-positive T cells) and then detec-ted by immunoblot assay to analyze the changes in the expression of cGAS .A hemagglutinin ( HA)-tagged cGAS plasmid was constructed and transfected into HeLa cells .Twenty-four hours after transfection , these cells were co-cultured with MT2 cells for another 24 hours.Immunoblot assay was used to detect the expres-sion of HTLV-1 proteins Tax and p19.Real-time PCR was performed to measure the expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level .Immunoblot assay was also used to analyze the phosphorylation of interferon regulatory factor 3 (IRF3) and p65.Expression of interferon (IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ) , RANTES ( regulated on activation , normal T cell expressed and secreted ) and tumor necrosis factor (TNF)-αwas detected by real-time PCR assay.Results Expression of cGAS was enhanced in HeLa cells after co-cultured with MT2 cells.Compared with control cells , the HeLa cells that were trans-fected with cGAS plasmid showed lower levels of Tax and p 19 proteins, suppressed expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level , enhanced phosphorylation of IRF 3 and p65, and higher levels of IFN-β, IP-10, RANTES and TNF-αafter co-cultured with MT2 cells.Conclusion cGAS might promote the innate immune response and inhibit HTLV-1 replication in HTLV-1-infected HeLa cells .

Role of NO/cGMP signaling pathway in dexmedetomidine-induced reduction of neuropathic pain in rats / 中华麻醉学杂志

Objective To investigate the role of nitric oxide/cyclic guanosine monophosphate (NO/ cGMP) signaling pathway in dexmedetomidine-induced reduction of neuropathic pain (NP) in the rats.Methods Forty-two healthy male Sprague-Dawley rats,weighing 200-250 g,were randomly divided into 7 groups (n =6 each) using a random number table:normal control group (C group);NP group;dexmedetomidine group (D group);dimethyl sulfoxide (DMSO) group;a non-selective nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) group (L-NAME group);inactive enantiomer D-NAME group (D-NAME group);a soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxidazole[4,3-a] quinoxalin-1-one (ODQ) group (ODQ group).NP was induced by chronic constriction injury to the sciatic nerve in anesthetized rats.On day 7 after chronic constriction injury,dexmedetomidine 1.5 μg/kg was injected intrathecally in group D,and in DMSO,L-NAME,D-NAME and ODQ groups,DMSO l0 μl,L-NAME 100 μg,D-NAME 100 μg and ODQ 10 μg were injected intrathecally,respectively,and 25 min later dexmedetomidine 1.5 μg/kg was injected intrathecally.The thermal paw withdrawal latency was measured immediately after intrathecal administration and at 30,60,90,120,150,180 and 210 min after intrathecal administration,and the area under the curve of thermal paw withdrawal latency was calculated to reflect the thermal pain threshold.Results Compared with group C,the thermal pain threshold was significantly decreased in the other six groups (P＜0.05).The thermal pain threshold was significantly higher in D,DMSO,L-NAME,ODQ and D-NAME groups than in group NP (P＜0.05).Compared with group D,the thermal pain threshold was significantly decreased in L-NAME and ODQ groups (P＜0.05),and no significant change was found in the thermal pain threshold in D-NAME and DMSO groups (P＞0.05).Conclusion The mechanism by which dexmedetomidine reduces NP is related to activation of NO/cGMP signaling pathway in the rats.

NO-cGMP-PKG pathway and cardiovascular disease / 国际儿科学杂志

Nitric oxide ( NO ) , cyclic guanosine 3′, 5′-monophosphate ( cGMP ) and cGMP-dependent protein kinase( PKG) play an important role in a variety of tissue cells. This paper reviews the related literatures about the NO-cGMP-PKG pathway and the cardiovascular diseases since the close relationship between them by summarizing the founction of the NO-cGMP-PKG pathway and the affection on the cardiovascular diseases by the NO-cGMP-PKG pathway,aiming to find a new research direction of the pathogenesis of cardiovascular disea-ses.

Role of NO/cGMP in the cardioprotective effects of intrathecal morphine preconditioning against myocardial ischemia-reperfusion injury in rats / 中国药理学通报

Aim To investigate the role of NO/cGMP in the cardioprotective effects of intrathecal morphine preconditioning against myocardial ischemia-reperfu-sion injury in rats. Method 54 Male Sprague-Dawley Rats were used to establish the model of intrathecal catheter placement. The rats were randomly assigned to 9 groups. SHAM (sham group), CON (control, sa-line) , ITMP ( intrathecal morphine preconditioning, 3μg·kg-1 ) , L-NAME+ITMP ( NO synthetase inhibi-tor,L-NAME ) , ODQ + ITMP ( guanylate cyclase in-hibitor, ODQ ) , KT5823 + ITMP ( PKG inhibitor, KT5823),L-NAME,ODQ,KT5823,6ratsineach group. ITMP were produced by three cycles of 5 min intrathecal injection of morphine and 5 min intermis-sion before myocardial ischemia, CON were achieved by intrathecal injection of saline in the same way, L-NAME+ITMP, ODQ +ITMP, KT5823 +ITMP were prepared by intrathecally administering L-NAME ( 30 nmol), ODQ(11 nmol) and KT5823(20 pmol) 10 minutes prior to ITMP respectively, L-NAME, ODQ, KT5823 worked as the control of inhibitors themselves respectively without ITMP. Subsequently, all rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion except the SHAM group. Myocardial infarct size, as a percentage of the AAR, was determined by 2 , 3 , 5-triphenyltetrazolium stai-ning. Results Compared with CON, the volumes of IS and IS/AAR were reduced in ITMP ( P <0.01 );the protective effects of ITMP were abolished by pre-treatment with L-NAME, ODQ and KT5823 ( P <0.01 );Conclusions NO/cGMP might be involved in the cardioprotective effect of intrathecal morphine pre-conditioning against myocardial ischemia and reperfu-sion injury in rats.

Role of NO-cGMP-PKG signal transduction pathway in mitigation of myocardial ischemia-reperfusion injury by intrathecal morphine postconditioning in rats / 中华麻醉学杂志

Objective To evaluate the role of nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signal transduction pathway in mitigation of myocardial ischemia-reperfusion injury by intrathecal morphine postconditioning in rats.Methods Forty-eight male Sprague-Dawley rats in which intrathecal catheters were successfully placed without complications,weighing 250-350 g,were randomly assigned into 8 groups (n =6 each):normal saline group (NS group),morphine postconditioning group (Mp group),1-NG-nitroarginine methyl ester (L-NAME,NO synthase inhibitor) + morphine postconditioning group (L-NAME + MP group),ODQ (guanylate cyclase inhibitor) + morphine postconditioning group (ODQ + MP group),KT5823 (PKG inhibitor) + morphine postconditioning group (KT5823 + MP group),L-NAME group,ODQ group and KT5823 group.Myocardial ischemia was induced by 30 min of occlusion of anterior descending branch of left coronary artery followed by 2 h of reperfusion.At 25 rin of ischemia,normal saline 10 μl was intrathecally infused over 5 min in group NS,and morphine (3 μg/kg,10 μl) was intrathecally infused over 5 min in group MP.L-NAME (30 nmol,10 μl),ODQ (11 nmol,10 μl) and KT5823 (20 pmol,10 μl) were intrathecally injected at 10 rin before morphine postconditioning in L-NAME + MP,ODQ + MP and KT5823 + MP groups,respectively.Before myocardial ischemia (T0),at 25 and 30 min of ischemia (T1-2),and at 120 min of reperfusion (T3),MAP and HR were recorded,and rate-pressure product (RPP) was calculated.The rats were sacrificed at T3,and myocardial specimens were obtained for determination of myocardial infarct size as a percentage of area at risk (IS/AAR).Results MAP,HR and RPP were significantly lower at T1-3 than at T0 in each group.Compared with group NS,MAP was significantly increased at T3,and IS/AAR ratio was decreased in MP group,and no significant changes were found in the other groups.Compared with group MP,IS/AAR ratio was significantly increased in L-NAME + MP,ODQ + MP and KT5823 + MP groups,and no significant changes were found in the other groups.Conclusion NO-cGMP-PKG signal transduction pathway plays an important role in mitigation of myocardial ischemia-reperfusion injury by intrathecal morphine postconditioning in rats.

Dendroaspis natriuretic peptide regulates the cardiac L-type Ca2+ channel activity by the phosphorylation of alpha1c proteins

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.

Cinnamyl alcohol attenuates vasoconstriction by activation of K+ channels via NO-cGMP-protein kinase G pathway and inhibition of Rho-kinase

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 microM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), methylene blue (MB; 10(-5) M) and 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one, (ODQ; 10(-6) or 10(-7) M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10(-8) or 10(-9) M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K+ channel inhibitor glibenclamide (Gli; 10(-5) M) and the voltage-dependent K+ channel inhibitor 4-aminopyridine (4-AP; 2 x 10(-4) M). These results suggest that CAL induces vasorelaxation by activating K+ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.

Mechanisms of endothelial cell protection by curcumin in hypercholesterolemia.

Mechanism of curcumin for protection of endothelial cell was studied in cholesterolfed rabbits. Thirty rabbits were randomly divided into five groups. The negative control group was fed a standard diet, the positive control group was fed the same diet with 2 % cholesterol , the curcumin group was fed the same diet with 2 % cholesterol and curcumin 100 mg/Kg BW/day, 200 mg/Kg BW/day or 400 mg/Kg BW/day. The cholesterol-rich diet significantly increased Malondialdehyde (MDA) in the aortic blood vessels, as reflected by Thiobarbituric Acid-Reactive Substances (TBARS), inhibited endothelium-dependent vascular relaxations to acetylcholine and decrease cyclic GMP were compared with vessels from normal rabbits (negative control). In cholesterol-fed rabbits, curcumin treatment decreased MDA in plasma production, improved endothelium - dependent relaxations to acetylcholine and increase cyclic GMP production. These results suggest that dietary treatment of rabbits with curcumin may prevent superoxide anion (O2-) induced inactivation of endothelium-dependent relaxing factor (EDRF), improve the endothelium-dependent relaxation to acetylcholine in the aortic blood vessels and increase cyclic GMP content in aortic of cholesterol-fed rabbits.

Effects of sildenafil on the viability of random skin flaps / Os efeitos do sildenafil na viabilidade de retalhos cutâneos randômicos

PURPOSE: To assess the viability of McFarlane skin flaps in rats with administration of sildenafil. METHODS: Twenty Wistar rats were distributed into two groups: Control (dorsal skin flap, subdermal application of saline solution at 0.9 percent) and Study (dorsal skin flap, subdermal application of sildenafil). Seven days after the surgery, flaps were photographed and graphically rendered. Then, they were analyzed with AutoCAD software. Three biopsies (proximal, medial and distal) of each flap were collected for histological analysis. RESULTS: Macroscopic analysis showed that animals of the study group had greater necrotic areas (p=0.003) in the dorsal skin flaps. Additionally, histological analysis of the distal third of these flaps showed a tendency to less granulated tissue formation in animals treated with sildenafil. CONCLUSION: Sildenafil subdermally was associated with lower viability of the random skin flap in rats.

OBJETIVO: Avaliar a viabilidade de retalhos cutâneos de ratos à McFarlane após a administração de sildenafil. MÉTODOS: Vinte ratos Wistar foram distribuídos em dois grupos: Controle (confecção do retalho cutâneo dorsal, aplicação subdérmica de solução salina a 0,9 por cento) e Estudo (confecção do retalho cutâneo dorsal, aplicação subdérmica de sildenafil). Sete dias após a operação, os retalhos foram fotografados e representados graficamente, para serem analisados com o programa AutoCad. Três biópsias (cranial, média e caudal) foram coletadas de cada retalho, para análise histológica. RESULTADOS: A análise macroscópica evidenciou que os animais do grupo Estudo apresentaram maiores áreas de necrose (p=0,003) nos retalhos cutâneos dorsais. Além disso, a análise histológica dos terços distais dos retalhos mostrou uma tendência à formação de menos tecido de granulação nos animais que receberam o sildenafil. CONCLUSÃO: O sildenafil subdérmico esteve associado com uma pior viabilidade tecidual dos retalhos cutâneos dorsais de ratos.

Direct Effect of Carbon Monoxide on Relaxation Induced by Electrical Field Stimulation in Rat Corpus Cavernosum

PURPOSE: Carbon monoxide (CO) may mediate smooth muscle relaxation in the rat corpus cavernosum smooth muscle (CCSM). We hypothesized that CO plays a role in neurally derived, frequency-dependent relaxation of rat CCSM. MATERIALS AND METHODS: To study the effect of CO on CCSM relaxation induced by electrical field stimulation (EFS), a CCSM bundle was mounted on a force transducer and perfused with Hanks' balanced salt solution at 37degrees C with 95% O2 and 5% CO2. After 1 hour equilibration with -500 mg of passive tension, contraction of the CCSM bundle was elicited by 10(-5) M phenylephrine, which was continuously added with different concentrations of CO (1%, 2%, and 5%). Frequency-dependent relaxation was induced by EFS trains (0.2 ms at 0.5-32 Hz, for 10 s) repeated at 2 min intervals over 15 min in the presence of adrenergic and muscarinic receptor blocking agents (guanethidine and atropine, respectively). To study the distribution of heme oxygenase-2 (HO-2) in the rat CCSM, we performed immunohistochemical evaluation. RESULTS: CO produced a dose-dependent enhancement of EFS-induced relaxation. Pretreatment with N(G)-nitro-L-arginine (a nitric oxide synthase blocker) greatly reduced the EFS-induced relaxation in the presence of CO (-45%). Pretreatment with zinc protoporphyrin-IX (ZnPP-9, a heme oxygenase inhibitor) had no significant effect on EFS-induced relaxation in the absence or the presence of CO. We found immunoreactivity for HO-2 in CCSM and immunoreactivity for protein gene product 9.5 (PGP 9.5) in nerve fibers. CONCLUSIONS: We conclude that CO produced a dose-dependent enhancement of EFS-induced relaxation in rat CCSM bundles, but neurally derived, frequency-dependent relaxation in the rat CCSM depended mostly on nitric oxide in response to nonadrenergic noncholinergic neurotransmission. Immunoreactivity for HO-2 was found in rat CCSM but not nerve fibers.

Effect of electro-acupuncture on NO-cGMP signal pathway in spinal cord in a rat model of neuropathic pain / 中华麻醉学杂志

Objective To investigate the effect of electro-acupuncture on the spinal nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal pathway in a rat model of neuropathic pain (NP). Methods Forty-eight pathogen-free male SD rats weighing 190-210 g were randomly divided into 3 groups (n = 16 each):group Ⅰ sham operation (group S); group Ⅱ group NP and group Ⅲ electro-acupuncture + NP (group E). NP was induced by chronic constrictive injury (CCI). Right sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 chromic catgut. In group E Huantiao and Weizhong points on the operated side were stimulated with electric stimulator (frequency 2 Hz, wave length 0.6 ms, starting at a voltage of 1mA and increasing by 1 mA every 10 min) for 30 min once a day at 11-17 d after CCI. Pain threshold to mechanical and thermal nociceptive stimuli was measured before (T0 , baseline) and at 10 and 16 d after CCI (T1, T2). The animals were sacrificed at 17 d after CCI and the lumbar segment (L4-6) was removed for determination of the activities of total NO synthase (tNOS), induced and neural NOS (iNOS, nNOS) (by spectrophotometry), NO content (by nitrate reductase method) and cGMP content (by immuno-histochemistry) in the spinal cord in 8 animals and the expression of iNOS and nNOS in the dorsal horn of the spinal cord (by immuno-histochemistry) in another 8 animals in each group. Results CCI significantly decreased the mechanical and thermal pain threshold at T1 and T2 as compared with the baseline at T0 in group NP and E. Hyperalgesia induced by CCI was significantly attenuated by electro-acupuncture at T2 in group E as compared with group NP.CCI significantly increased tNOS and nNOS activities, NO and cGMP content in the spinal cord and up-regulated nNOS expression in the spinal dorsal horn. Electro-acupuncture significantly attenuated the CCI-induced increases in tNOS and nNOS activities, NO and cGMP content in the spinal cord and nNOS expression in the spinal dorsal horn. There was no significant difference in the iNOS activity among the 3 groups. Conclusion NO-cGMP signal pathway in the spinal cord is involved in the acupuncture analgesia.

Effects of lactoferrin on activity of PKG in spinal dorsal horn in a rat model of neuropathic pain / 中华麻醉学杂志

Objective To investigate the effects of lactoferrin on activity of PKG in spinal dorsal horn in a rat model of neuropathic pain(NP).Methods Thirty-two male SD rats weighing 200-250 g were randomly divided into4 groups(n = 8 each): sham operation group(group S),NP group,lactoferrin group and KT5823(an inhibitor of PKG)group.Neuropathic pain was produced by placing loosely constrictive ligatures around the common sciatic nerve in group NP,lactoferrin and KT5823,while the sciatic nerve was only exposed but not ligated in groupS.In group S and NP,normal saline 10 μl + 50% dimethyl sulfoxide(DMSO)10 μl were injected intrathecally.Lactoferrin 100 μg + 50% DMSO 10 μl were given intrathecally in group lactoferrin.Lactoferrin 100 μg + KT5823 10 μl were given intrathecally in group KT5823.The paw withdrawal latency(PWL)to a thermal nociceptive stimulus was measured every 30 min within 180 min after administration.The rats were then sacrificed and the spinal cord was removed.The activity of PKG in the spinal dorsal horn was determined by immunofluorescence.Results Compared with group NP and KT5823,the PWL was significantly prolonged after administration in group lactoferrin and the PKG activity was significantly increased in group lactoferrin(P ＜ 0.05).There was no significant difference in the parameters mentioned above between group NP and group KT5823(P ＞ 0.05).Conclusion Lactoferrin reduces NP by inhibiting the activity of PKG in spinal dorsal horn in rats.

Preventive Effect of Pentoxifylline on Cyclosporine A-Induced Collagen Synthesis in Calf Pulmonary Artery Endothelial Cells

PURPOSE: Cyclosporine A (CsA) is a potent immunosuppressive agent, and it has been used to prevent rejection of transplanted organs and to treat autoimmune diseases. Many side effects of CsA, including various types of endothelial dysfunction, have been reported. Pentoxifylline (PTX) is a non-selective phosphodiesterase inhibitor that is used for the treatment of peripheral vascular diseases. METHODS: We investigated the effect of CsA on collagen synthesis and clarified whether PTX has protective effects against CsA-induced arterial vasculopathy using calf pulmonary artery endothelial cells. This study was carried out using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription- polymerase chain reaction (RT-PCR), Western blot analysis, nitric oxide (NO) detection, and cyclic guanosine monophosphate (cGMP) enzyme immunoassay. RESULTS: CsA treatment significantly increased the expression of collagen type I mRNA and protein and decreased the production of NO and cGMP. However, pre-treatment with PTX exerted anticollagen effect by suppressing the CsA-induced formation of collagen, but this effect of PTX was not modulated by NO and cGMP. CONCLUSION: Based on the present results, it is expected that PTX may have a protective effect against CsA-induced arterial vasculopathy, although the mechanism of PTX needs to be clarified in future studies.