| Endotoxic shock (ES) is mostly caused by Gram-negative bacteria and characterized by hypotension through vascular regulation dysfunction and organ damage through whole body inflammation. The main active component of endotoxin, lipopolysaccharide (LPS), may stimulate many endogenous inflammatory agents such as complement, kinin and many cytokines which include tumor necrosis factor (TNF-α), interleukin-1β(IL-1β) and interferon-γ(IFN-γ) etc. It has been established that maybe inflammatory factors themselves are vascular active component, which is crucially involved in the pathogenesis of endotoxic shock. However, the mechanism of development of inflammatory damage in organ failure during endotoxic shock is still not yet known, and further, it is more difficult in clinical treatment. In recent years, some molecular biology study has found that some small gas molecus such as nitric oxide (NO) and carbon monoxide (CO) are increased during shock and may play an important role in the regulation of blood pressure and vascular relaxation. Although the mechanism of hypotension during endotoxic shock is still not established yet, many studies have shown that, increased NO and CO may play"double–side"role in the development of severe hypotension during endotoxic shock. Selective inhibition of NO production by may reduce inflammtion-casued tissue damage during endotoxic shock, however, although total inhibition of production of NO and CO may increase blood pressure in endotoxic shock animals, the mobility and mortality are not altered, which might be even worse. In the middle of 90's, it has been found that hydrogen sulfide (H2S) is one of the metabolite of aminothiopropionic acid, which might regulate the function of nervous system, especially hippocamp, and might regulation the tension of digestive tube and vascular, which has the similar biological function with NO and CO. Recently, major concern has emerged that H2S might be a novel endogenous agent during the patho-physiological reactions during endotoxic shock. Until now, the role of H2S playing in endotoxic shock is still quite confusing. It has been reported that increased production of H2S may induce hypotension and tissue inflammatory damage and these can be alleviated by H2S inhibition. While, some studies suggested that H2S might have protective effects in tissue, eg. Yuka et al has reported endogenous H2S may protect neuron from oxidative damage through increasing the production of glutathione. Study from Bian et al has also shown that H2S might be protective in cardiac hypoxia damage. Also from the study of Fiorucci et al, it has been suggested that H2S may reduce stomach damage caused by anti-inflammatory nonsteroidal drugs through improve its micro-circulation.Therefore, this study is established to explore the relationship between the concentrations of H2S in serum and hypotension and tissue inflammatory damage, which are on the basis of rat endotoxic shock model. From this research, the alteration of anti-inflammatory nonsteroidal drugs, the function of H2S and its relationship with NO and CO during endotoxic shock, are all studied, which may disclose the pathogenesis of endotoxic shock.1 Establishment of rat endotoxic shock modelEndotoxic shock is a widely seen severe clinical disease characterized by acute micro-circulation failure. Therefore, how to prevent and cure endotoxic shock is a big issue that needs to be concerned. LPS is the primary cause for endotoxic shock. In the present study, we have established animals model of endotoxic shock through injection of LPS.64 male Wistar rats were randomly divided into 3 groups: 1. control group (n=18): Rat were inject with saline (1 ml/kg); 2. small dose LPS group (n=22): rats were inject with low dose LPS 5 mg/kg (LPS 5 mg/ml, 1 ml/kg); 3. large dose LPS group (n=24): rats were injected with LPS 10 mg/kg (LPS 10 mg/ml, 1 ml/kg). Mean artery pressure and heart rate were continuously examined in 6 hrs after LPS injection. Serum samples were taken at different time point in all 3 groups (30, 60, 90 min, 2, 3, 4, 6 hrs). Plasmas were stored until the concentrations of TNF-αwere measured. Survival was calculated in all groups after the experiment.Results: 1. There were no differences in basic blood pressure among all 3 groups before treatment (P > 0.05). After the treatment with saline or LPS, there was no difference in MAP in control animals, which was maintained between 110±5 mmHg ~106±9 mmHg. MAP was rapidly reduced in large dose LPS treated animals, however, this alteration can be normalized in 15 min and sustained for about 60 min and then reduced slowly and continuously till the lowest level (69±4 mmHg) at 4 hrs,and this may maintain till 6 hrs. The blood pressure was significantly lower in large lose LPS treated group compared with same-time point saline-treated controls (all P < 0.01). There was no big alteration in MAP in small dose of LPS-treated animas in first 3 hrs after treatment, and then, MAP was decreased slowly at 3hr time and rapidly at 4 hrs time point. The lowest level was reached at 6 hrs. 2. The heart rate was transiently decreased after large dose of LPS treatment and then increased to normal level that sustained for about 30 mins. Heart rates were decreased slowly from 90-120min until animal died. In addition, animals with big increase of hear rate may have longer survival time, while no increase in heart rate may correlate with less survival. Transient increase and then decrease in heart rate were found in control and small dose LPS treated animals. 3. No death existed in saline-treated control group. The survival rate of animals with treatment of large dose of LPS was 66.67% (16/24,P < 0.05 vs control), while small dose of was 90.91% (20/22). The minimum survival time in rats was 3.5 hrs. 4. Concentrations of TNF-αwere significantly increased in both large and small dose LPS treated animals compared with controls (all P < 0.05 vs. control), magnificently increased at 60 min (all P < 0.05 vs. control), and arrived peak at 90 min(all P < 0.01 vs. control), then decrease until normal level at about 3 hr (all P > 0.05 vs. control). The trend of TNF-αincreasing is bigger than large dose LPS treated group. TNF-αcould not be found in control animals at any time point. These results suggest that this animal model established by LPS injection is successful and ideal. The alteration of MAP, heart rates in these animal models are similar with those found in inflammation-caused shock2 Concentrations of H2S in serum and organs in rat endotoxic shock model and its meanings2.1 Concentrations of H2S in serum in rat endotoxic shock modelEndotoxic shock is mostly caused by LPS and clinically characterized by vascular regulation dysfunction, vascular dilation and hypotension. Recently, it has been suggested that NO and CO are increased during endotoxic shock and might play important role in regulation of blood pressure and vascular dilation. However, the mechanism of hypotension during endotoxic shock is still not yet known. Large amount of data suggest that except large amount production of NO and CO, H2S might be a novel endogenous agent during the patho-physilological reactions during endotoxic shock. Until now, the role of H2S playing in endotoxic shock is still quite confusing. In the present study, we have explored the effect of H2S on endotoxic shock through observation of serum H2S concentrations.96 male Wistar rats were divided into 4 groups (n=24 in per group): 1. Control group: animals were received saline (1 ml/kg); 2. LPS group: rats were injected with LPS (10 mg/ml, 1 ml/kg); 3. LPS+NaHS (H2S provider) group: animals were injected with NaHS (28μmol/kg in 0.5 ml saline) i.p 10min before the injection of LPS. 4. LPS+ PPG (H2S inhibitor) group: animals were injected with PPG (45 mg/kg in 0.5 ml saline) i.p 30 min before LPS injection. MAP and heart rate were continuously examined in 6 hrs after treatment and plasma were taken to for measurement of H2S at different time point (30, 60, 90 min, 2, 3, 4, 6 hrs).Results: 1. there were no differences in the basic blood pressure between rats of 4 groups (P > 0.05). There was no alteration in MAP in control animals in 6h experiment period which maintain between 110±5 mmHg ~106±9 mmHg. MAP was rapidly decreased in LPS-treated rats, but returned to normal level in 15min, which sustained for about 60 min and then decreased slowly. At 90 min (81±3 mmHg), MAP was significantly and continuously decreased, and the lowest level were reached at about 4 hrs (69±4 mmHg) and this level were sustained till 6 hrs. MAP in this group was lower compared with control group at the same time point (all P < 0.01). MAP at 90 min, 4 hrs and 6 hrs were 97±5 mmHg, 92±6 mmHg and 88±7 mmHg separately, all significantly higher than ES group at the same time point (P < 0.05 or P < 0.01). Compared with endotoxic shock group, MAP were lower at 90 min, 4 hrs and 6 hrs (P < 0.05 or P < 0.01), which were 74±6 mmHg, 51±5 mmHg and 43±4 mmHg separately. 2. Heart rates of LPS treated rats were transiently decreased and then increased until normal level, which was sustained for about 30 min. The heart rate of this LPS-treated rats were slowly decreased from 90~120 min and then animal died. The heart rate of LPS+NaHS-treated animals was significantly reduced compared with same time point LPS group (3, 4, 6 hrs,P < 0.05). That heart rate was transiently increased and then decreased were found in both control and LPS+PPG groups. 3. Concentration of H2S in plasma was tended to increase in LPS-treated animals. Significantly increased H2S levels were found in 4 hrs and 6 hrs (all P < 0.05), and yet, plasma H2S concentrations (4 hrs and 6 hrs after LPS treatment) in LPS-treated animals was negatively correlated with blood pressure and the coefficient correlation was -0.973 and -0.986 respectively (all P < 0.05); Compared with LPS treatments, plasma H2S concentrations in LPS+PPG group were significantly lower at 4 hrs and 6 hrs after treatment (P < 0.05), while significantly increased in LPS+NaHS group (4, 6 hrs after LPS injection) (all P < 0.05).All these results suggested that hypotension in endotoxic shock is partly induced by increase of H2S.2.2 Concentrations of H2S in organs in rat endotoxic shock modelOur data have shown that hypotension in endotoxic shock is partly induced by increase of H2S. In this part of study, we have further investigated the effect H2S on endotoxic shock through observation of H2S concentration in main organs. 154 male Wistar rats were seldomly divided into 4 groups: 1. control group (n=28): animalswere injected with saline (1 ml/kg). 2. LPS group (n=50): animals were received LPS (10 mg/kg, 10 mg/ml); 3. LPS+ NaHS (H2S provider) group (n=38): animals were injected with NaHS i.p 10min before LPS injection(28μmol/kg in 0.5 ml saline); 4. LPS+ PPG (H2S inhibitor) group (n=38): animals were received PPG (45 mg/kg in 0.5 ml saline) i.p 30min before LPS injection. Serum, plasma, and spleen were collected at different time point (0 hrs, 2 hrs, 4 hrs, 6 hrs) and heart, liver, lung and kidney were taken at 6 hrs. Malondialdehyde (MDA)concentration, Myeloperoxidase (MPO) activity, hydrogen sulfide synthase activity and H2S concentration were determined. Lactate dehydrogenase (LDH), alanine aminotransferase (ALT) in serum, urea nitrogen (BUN)and creatinine (Cr) levels in plasma, IL-1βand IL-6 concentrations in spleen tissue were determined. PaO2 was measured and morphological alterations were examined.Results: 1. MDA content and MPO activity were all significantly increased in all organs in LPS-treated animals compared with controls at 6 hrs(all P < 0.01). Compared with LPS-treated group, MDA content and MPO activity were all significantly lower in LPS+PPG group(all P < 0.01), while higher in LPS+NaHS group (all P <0.01). 2. Serum LDH and ALT activity were higher in LPS group compared with controls (all P < 0.05). Compared with LPS–treated animals, LDH and ALT activity in serum were significantly reduced in LPS+PPG-treated rats (all P < 0.05) while higher in LPS+NaHS group (all P < 0.05). 3. Concentration of IL-1βin spleen tissue was too low to be detected in control animals, and the concentration of IL-6 was 54.03±6.57 ng/L. Concentrations of cellular factors in rat spleen tissue was increased following the time of LPS treatment and were all significantly different at all time point compared with control animals (all P <0.05). Concentrations of IL-1βand IL-6 may reach the peak at 2 hrs and 4 hrs after LPS treatment, which were 342.14±14.89 ng/L and 4022.76±39.72 ng/L separately. Concentrations of IL-1β(2 hrs) and IL-6 (4 hrs) in LPS+PPG group were reduced to 86.9%, 62.8% (all P < 0.05) compared with LPS group at the same time point, but were still higher than controls (all P < 0.05 vs. control). IL-1β(2 hrs) and IL-6 (4 hrs) concentrations in spleen of LPS+NaHS–treated animals were increased to 86.9% and 62.8% separately compares with LPS–treated animals at the same time point (all P < 0.05). 4. PaO2 was significantly higher in rats pretreated with PPG and lower in rats pretreated with NaHS compared with LPS groups(all P < 0.05 ). 5. There were no differences in plasma BUN and Cr concentration in all groups before treatment, while LPS-treatment may significantly increase plasma BUN and Cr at 2, 4 and 6 hrs (all P < 0.05). Compared with LPS-treated group, LPS+PPG treatment led to significantly reduced plasma BUN and Cr content compared (all P <0.05), while LPS+NaHS treatment may significantly increase plasma 4 h,6 h BUN and 2h,4h Cr content (all P < 0.05). 6. LPS treatment led to significantly increased H2S content and hydrogen sulfide synthase activity in rat tissue compared with control animals (all P < 0.05). Compared with LPS-treatment animals, H2S content and hydrogen sulfide synthase activity were significantly decreased in LPS+PPG treated group (all P < 0.05) while increased in LPS+NaHS treated animal (all P < 0.05). 7. The morphology is normal in control animals. LPS injection may cause clear tissue damage that was characterized by celluar swollen, and inflammatory cells accumulation. This damage could be ameliorated by PPG and can be worsen by NaHS.Results suggested that increased H2S production partly participated in rat tissue inflammatory damage during endotoxic shock.3 The effect of H2S in endotoxic shock and its relationship with NOAs our described previously, increased production of endogenous H2S production may partly participated in rat tissue inflammatory damage during endotoxic shock. The similar experiment conducted in our lab have shown that, in the model of acute lung injury induced by LPS, reduced production of endogenous H2S and increased NO might be the reason. Exogenous H2S may protect tissue from injury which might be due to its effect of inhibition of iNOS activity and reduce NO production. However, so far the relationship between H2S and NO in endotoxic shock is still confusing. In this study, we administered propargylglycine (PPG, H2S inhibitor) and NaHS (exogenous H2S provider) to endotoxic shock rat model, to check the effect of H2S on NOS/NO system.32 male Wistar rats were seldomly divided into four groups: 1. Control group: animals were treated with saline (1 ml/kg). 2. LPS group: animals were received with LPS (10 mg/kg, 10 mg/ml); 3. LPS+ NaHS (H2S provider) group: rats were injected with NaHS (28μmol/kg in 0.5 ml saline) i.p. 10 min before LPS injection. 4. LPS+ PPG (H2S inhibitor) group:rats were injected with PPG (45 mg/kg,in 0.5 ml saline ) i.p. 30min before LPS injection。Some organs such as heart, liver, spleen, lung and kidney were collected at 6 hrs. NOS activity and NO content in tissue were determined by kits. iNOS protein expression in tissues were checked by immunohistochemistry and Western blot.Results have indicated that: 1. iNOS activity and NO content were significantly increased in LPS treated group compared with controls (P < 0.05 or P < 0.01). Compared with LPS treatment, iNOS activity and NO content were significantly decreased in LPS+PPG group and increased in LPS+NaHS in tissues(P < 0.05 or P < 0.01). 2. Activity of eNOS in tissues was significantly lower in LPS treated animals compared with controls (all P < 0.05). Compared with LPS treated animals, Activity of eNOS was significantly increased in LPS+PPG-treated rats (all P < 0.05) while decreased in NaHS+LPS groups (all P < 0.05). 3. Immunostaining for iNOS showed that there is no iNOS protein positive expression in tissue of control animals. However, LPS may stimulate iNOS expression in cells which were mainly distributed in intracytoplasm of inflammatory cells, all parenchymal cell and vascular smooth muscle cell. LPS induction of iNOS expression can be reduced by PPG and enhanced by NaHS. 4. Western blot for iNOS showed that LPS may increase iNOS protein (all P < 0.05) in all tissues and this effect can be amlierated by PPG (P < 0.05 or P < 0.01) and enhanced by NaHS (all P < 0.05). These results suggested that H2S may decrease the activity of eNOS and increased the activity of iNOS, which may result in large production of NO. This effect might cause tissue damage during endotoxic shock.4 The effect of H2S in endotoxic shock and its relationship with COStudies accumulated in recent years have showed that, the incidence of hypotensionand inflammatory damage in tissues might not be all achieved by NO that induced by iNOS, heme oxygenase-1/carbon monoxide ( HO-1/CO)might be also crucial as a endogenous agent and therefore get more attention. Our previous study has found that increased endogenous H2S level might be partly involved in the pathogenesis of hypotension and tissue damage. Therefore, all three endogenous gas molecule i.e. NO, CO and H2S are all involved in the incidence of ES. We already identified that the funtion of H2S might correlated with NO in the 3rd experiment. In the present experiment, we will check the effect of H2S on HO-1/CO.64 male Wistar rats were randomly divided into four groups: 1. control group: animals were treated with saline (1 ml/kg). 2. LPS group: animals were received with LPS (10 mg/kg, 10 mg/ml); 3. LPS+ NaHS (H2S provider) group: rats were injected with NaHS (28μmol/kg in 0.5 ml saline) i.p. 10 min before LPS injection. 4. LPS+ PPG (H2S inhibitor) group: rats were injected with PPG (45 mg/kg,in 0.5 ml saline)i.p. 30min before LPS injection. Anticoagulant blood were collected from femoral artery at different time point (0, 30, 60, 90 min, 2, 3, 4, 6 hrs) and some organs such as heart, liver, spleen, lung and kidney were collected at 6 hrs. CO concentraton in blood and all tissues were determined and HO-1 protein expression in tissues were checked by immunohistochemistry and Western blot.Results have indicated that: 1. CO concentraton in blood and all tissues were significantly increased in LPS treated group compared with controls (all P < 0.05). Compared with LPS treatment, CO concentraton in blood and all tissues were significantly decreased in LPS+PPG group and increased in LPS+NaHS in tissues (all P < 0.05). 2. Immunostaining for HO-1 showed that there is no HO-1 protein positive expression in tissue of control animals. However, LPS may stimulate HO-1 expression in cells which were mainly distributed in intracytoplasm of inflammatory cells, all parenchymal cell and vascular smooth muscle cell. LPS induction of HO-1 expression can be reduced by PPG and enhanced by NaHS. 4. Western blot for HO-1 showed that LPS may increase HO-1 protein (all P < 0.05) in all tissues and this effect can be amlierated by PPG and enhanced by NaHS (all P < 0.05).Results have shown that H2S may up-regulate HO-1/CO in the tissue of endotoxic shock rat model. However, the function and mechanism of HO-1/CO up-regulation in endotoxic shock still needs to be further investigated.CONCLUSIONIn the present study, we have investigated the role of H2S in pathogenesis of endotoxic shock through its relationship with NO and CO in endotoxic shock rat model in whole and molecular level, which might provide some mechanism of endotoxic shock pathogenesis.1 Endotoxic shock animals model can be created by large dose of LPS injection. This animal model is ideal since the alterations in MAP, heart rate are similar with those found in sepsis shock.2 Increased H2S production might be partly involved in the pathogenesis of hypotension and tissue inflammatory damage during endotoxic shock.3 It is our first to find that H2S may cause decreased eNOS activity and increased iNOS activity and large production of NO, this may correlated with endotoxic shock and tissue inflammatory damage.4 It is our first to find that H2S may up-regulate HO-1/CO in the tissue of endotoxic shock rat model. However, the function and mechanism of HO-1/CO up-regulation in endotoxic shock is still needs to be further investigated.
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