| PartI Hydrogen Sulfide protects against cigarette smoking-induced leftventricular dysfunction in ratObjective:The present study was designed to investigate the protective effects ofhydrogen. sulfide(H2S) against cigarette smoking-induced leftventriculardysfunction in rats.Materials and methods1. Male Sprague-Dawley rats weighing200-250g were housed five per plastic cage inan animal room with an alternating12h light/dark cycle at23±2°C and50±5%relative humidity.During the first week, the number of cigarettes was gradually increasedfrom5to10cigarettes over a30-min period, twice in the morning and twice in theafternoon. After that,10cigarettes were used in each smoking trial, repeated4times/dayfor the rest of the16weeks experimental period.The rats were randomly divided into4groups: CS group (n=10; exposed to cigarette smoke at the rate of40cigarettes/day), NaHSgroup (n=10; administered intragastrically with NaHS solution at a dose of8μmol/kgonce daily in the morning), CS+NaHS group (n=10; exposed to cigarette smoke andadministrated with NaHS), and control group (n=10; neither exposed to cigarette smokenor treated with NaHS).2. The plasma and myocardial contents of H2S were measured by methylene blue andmodified sulfide electrode methods.Two-dimensional echocardiography was used toevaluate cardiac structure and function.The Annexin V-FITC apoptosis detection kit wasused to determine cardiomyocyte apoptosis.Annexin V abeled with a fluorophore couldidentify cells in the early stage of apoptosis,and propidine iodine (PI), afluorescent nucleicacid binding dye,was responsible for staining cells in the medium and late stages ofapoptosis.Cardiomyocyte apoptosis was evaluated with the TUNEL staining.PI3K activityin the immunocomplexes was measured using PI3K ELISA kit,phosphorylation of JNKand p38MAPK was measured by Western blot analysis. 3. All data analysis was performed with SPSS17.0Statisstical software,a P<0.05wasconsidered for quantification.Results:1. Compared to the control group,H2S contents in plasma and myocardial tissue weresignificantly lower in the CS group (P<0.05),while in the CS+NaHS group, H2S contentswere markedly higher than those in the CS group (P<0.05).2. Compared to the control group,LVEDD and LVESD were significantly increased inthe CS group(P<0.05),LVFS and LVEF were remarkably lower(P<0.05).However,Theabove four parameters were markedly higher in the CS+NaHS group than in the CSgroup(P<0.05).3. The apoptotic cardiomyocytes were significantly increased in the CS groupcompared to the control group(P<0.05),whereas apoptotic cells were remarkably decreasedin the CS+NaHS group compared to the CS group(P<0.05).4. Compared to the control group,Cytosolic cytochrome c(Cytc)、cleaved caspase-9and caspase-3、Bax/Bcl-2ratiowere significantly increased in the CS group(P<0.05), whilein the CS+NaHS group, the four were markedly decreased than the CS group(P<0.05).5. Compared to the control group,the protein levels of phospho-JNK and phospho-p38were significantly elevated in the CS group. Administration of NaHS remarkably inhibitedthe activation of MAPKs in the CS+NaHS group.6. Compared to the control group,the PI3K activity was remarkably lower in theCS,while in the CS+NaHS group(P<0.05),the PI3K activity was markedly higher than thatin the CS group(P<0.05).The protein level of phospho-Akt was reduced in the CS groupand elevated in the CS+NaHS group(P<0.05).Conclusions:H2S may exert anti-apoptotic effects in the myocardium of smoking rats by inhibitingJNK and p38MAPK pathways and activating PI3K/Akt signaling. Our study demonstratesthat H2S protects against cigarette smoking-induced left ventricular systolic dysfunction viaregulation of apoptosis. PartII Hydrogen sulfide protects against cigarette smoking-inducedpulmonary fibrosis in ratObjective:We established a rat model of passive smoking and investigated whether ornot H2S has protective effects against pulmonary fibrosis induced by chronic cigarettesmoke exposure.Materials and methods1. Male Sprague-Dawley rats weighing200-250g were housed five per plastic cagein an animal room with an alternating12h light/dark cycle at23±2°C and50±5%relative humidity.During the first week, the number of cigarettes was gradually increasedfrom5to10cigarettes over a30-min period, twice in the morning and twice in theafternoon. After that,10cigarettes were used in each smoking trial, repeated4times/dayfor the rest of the16weeks experimental period.The rats were randomly divided into4groups: CS group (n=10; exposed to cigarette smoke at the rate of40cigarettes/day), NaHSgroup (n=10; administered intragastrically with NaHS solution at a dose of8μmol/kgonce daily in the morning), CS+NaHS group (n=10; exposed to cigarette smoke andadministrated with NaHS), and control group (n=10; neither exposed to cigarette smokenor treated with NaHS).2. Rat lung tissues were stained with hematoxylin-eosin and Masson’s trichrome;Theexpression of type I collagen was detected by immunohistochemistry;Oxidative stress wasevaluated by detecting serum levels of malondialdehyde,superoxide dismutase andglutathione peroxidase and measuring the reactive oxygen species generation in lung tissue;Inflammation was assessed by measuring serum levels of inflammatory cytokines,including hs-CRP, TNF-α,IL-1βand IL-6;The protein expression of Nrf2, NF-κB andphosphorylated MAPKs in the pulmonary tissue was determined by Western blotting.3. All data analysis was performed with SPSS17.0Statisstical software,a P<0.05wasconsidered for quantification.Results:1. H2S levels in plasma and lung tissue were significantly lower in the CS group thanin the control group(P<0.05), while in the CS+NaHS group, H2S contents were remarkablyhigher than those in the CS group(P<0.05).2.Compared to the control group,pulmonary fibrosis and inflammatory cell infiltrationcould be observed,type I collagen was upregulated in the smoking rats(P<0.05). Interstitial fibrosis and inflammatory response of the lung tissue significantly attenuat，type Icollagen was downregulated in CS+NaHS group than in the CS group(P<0.05).3. Compared to the control group，MDA level was significantly elevated while SODand GSH-Px activities were reduced,ROS production was significantly elevated in the CSgroup(P<0.05),while MDA level was remarkably decreased and SOD and GSH-Pxactivities were increased,ROS production was markedly elevated in CS+NaHSgroup(P<0.05).4. Compared to the CS group,the protein levels of two downstream targets of Nrf2,HO-1and Trx-1, were remarkably elevated in the CS+NaHS group.5. In the CS group, the concentrations of hs-CRP, TNF-α, IL-1βand IL-6weresignificantly increased,serum levels of these cytokines were markedly decreased in theCS+NaHS group.6. In the CS group,The protein levels of phospho-ERK1/2, phospho-JNK andphospho-p38were significantly elevated,the phosphorylation of MAPKs was found toremarkably inhibited in the CS+NaHS group.7. In the CS group, the nuclear expression and NA-binding activity of NF-κB weremarkedly increased.The activation of NF-κB was found to significantly inhibited in theCS+NaHS group.Conclusion:H2S can increase nuclear localization of Nrf2in the lung tissue and consequentlyupregulate the expression of antioxidant genes HO-1and Trx-1in the smokingrats.Furthermore,H2S can also reduce cigarette smoking-induced inflammation byinhibiting the phosphorylation of ERK1/2, JNK and p38MAPK and negatively regulatingNF-κB activation.Our study demonstrates that H2S protects against pulmonary fibrosis inthe smoking rats via attenuation of oxidative stress and inflammation. |
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