| Background:Decompression sickness(DCS)describes a series of signs and syndromes arising from bubbles which coming out of dissolved gas inside the body on decreased atmospheric pressure.Those clinical manifestations range from mild as itchy skin to severe as abnormal circulatory and respiratory dysfunction,nervous system dysfunction,and even death.DCS is a common disease in diving career,and is of frequent occurence in military diving,such as submarine escape.This prompted us to conduct efforts in research of preventive medicine or the other means to DCS.It's not only the focus of diving medical research but also of particular concern to the health of military divers.It is confirmed that pulmonary vascular endothelial cell(PVEc)is the first target of decompressing intravascular bubbles,according to decompression theory of bubble dynamics and physiological function of circulatory system.Therefore,protection of endothelium is supposed to be beneficial to prevention of DCS by reduce decompression injury.Because the underlying mechanisms are obscure,single target drugs for endothelial cell protection do not achieve expected outcome.This suggested that decompression injury to endothelium was complicated and multichannel beyond effects of single target drugs.Traditional Chinese Medicine(TCM)is hoped to fulfill it with its feature of multitarget effects.We found in pre-experiment that berberine(BBR)may came into effect on account of its pharmacological mechanism in respond to decompression injury of vascular endothelium and its secondary reaction.This research is focus on:First,the effects of pre-conditioning with BBR on prevention of DCS in rats,and then explore the dose-dependent manner;Second,role of decompression stress on apoptosis of PVEc induced by decompression stress and protective effects of pre-conditioning with BBR;last,role of pulmonary endothelium-derived vasoactive factors in decompression stress and protective effects of pre-conditioning with BBR by MAPK-NOS-NO pathway.These efforts are hoped to further insight into the role of PVEc in decompression stress and highlight a new pharmacological intervention measure for DCS prevention.Methods:1.The subject rats were divided into control,model,BBR precondition groups.The latter included Low dose(LD)group,Medium dose(MD)group,High dose(HD)group,and Long-term medication(LT)group.Animal decompression damage model was prepared by hyperbaric exposure to 60msw depth of air with 60min bottom time,and then surface with approximately uniform pressure reduction in 30s.Method of BBR preconditioning was briefly described as follow:BBR pre-conditioning were administrated by intraperitoneal injection with distilled water solutions.a single dose for LD group(12.5mg/kg),MD group(25mg/kg)and HD group(50mg/kg)were administrated just 3h before hyperbaric exposure.For LT group,BBR is continuous administration for 3 days in a dose of 50mg/kg same as HD group with the last dose administrated just 3h before hyperbaric exposure.The same volume of physiological saline was used as pseudo-drug injected same time.Evaluating indicators of decompression damage and effects of drug pre-conditioning included:clinical manifestations of DCS with time of onset,time of death and mortality;estimation of lung tissue injury with histopathological section and tissue wet-dry weight ratio;differential blood count and platelet activity with whole blood automatic analyzer;the content of desmosine(DES)in Peripheral blood and IL-1?in lung tissue assayed by ELISA.2.For estimating effects of decompression damage alone or in combination with BBR pre-treatment on PVEc apoptosis and the role of NF-?B,evaluating indicators were more than the above decompression damage model evaluation index,including:quantitative analysis of cellular apoptosis factors such as B cell lymphoma/leukemia-2(Bcl-2),Bcl-2-related X protein(Bax),caspase-3 and nuclear factor-?B(NF-?B)in PVEc with ELISA;Transcriptional level of BAX,Bcl-2 and Caspase-3 mRNA with Real-Time PCR(RT-PCR).3.For estimating the role of MAPK-NOS-NO pathway in decompression damage and BBR pre-treatment effects,more indicators were induced:content of NO in peripheral blood with nitric reductase method;distribution of eNOS,iNOS and nNOS in lung tissue by immunohistochemical method;Protein expression of eNOS,iNOS,nNOS,and the phosphorylation levels of ERK,JNK and p38 determined by Western blot analysis.SPSS20.0 was performed to statistics the data.Metrological data showed as meanąstandard deviation(xąSD).?~2 test,independent sample t test and ANOVA were used to compare differences between groups.The relationship between indicators were analyzed with Pearson correlation and regression curve fitting.p<0.05 was considered to be statistically significant.Results:1.The results of model preparation showed that,DCS onset rapidly after surface,and followed with 50%mortality.Compared with the control group,pathological section showed obvious pulmonary interstitial edema and hyperemia in model animals;WBC,RBC and NEUT in peripheral blood increased significantly(p<0.01),while PLT decreased significantly(p<0.01);wet-dry ratio of lung tissue increased significantly(p<0.05),blood DES increased significantly(p<0.01),and IL-1?content in lung tissue increased significantly(p<0.01).2.The results of evaluation index for decompression damage after BBR intervention showed that,the mortality decreased to 40%in HD,LT group and 30%in the other drug intervention groups.The DCS onset of the drug intervention group was later than that of the model(p<0.05).Pathological sections showed that BBR had a certain alleviating effect on leukocyte infiltration and edema in pulmonary interstitium.Compared with the intervention results of each group,the degree of pulmonary interstitial edema in the LD group was lighter than that in the HD group,suggesting that the use of low-dose BBR may be more conducive to the reduction of lung injury caused by extreme decompression.Compared with the model,WBC,RBC and NEUT in peripheral blood were significantly lower(p<0.01 or p<0.05),but still higher than those in the control group(p<0.01 or p<0.05).The PLT of the single dose drug intervention groups were significantly higher than that of the model group(p<0.01),but still lower than that of control group(p<0.01).The lung tissue wet-dry ratios of LD and MD group were not different from that of model but higher than that of control(p<0.01).DES of the intervention group was significantly lower than that of the model group(p<0.01,with LD group and HD group).Contents of IL-1?in the intervention groups'lung tissues were significantly lower than that of the model group(p<0.01).Summary of multiple evaluation results suggested that different doses of 3h pretreatment of BBR had certain effects on reducing body decompression injury,among which the LD group had better effects.There was no significant difference in the effect between the long-term drug use and the 3h drug use.3.Apoptosis-related proteins in pulmonary vascular tissue results showed that decompression injury significantly decreased the protein expression of Bcl-2 and increased the protein expression of Bax,decreased the Bcl-2/Bax andincreased the protein expression of Caspase-3(p<0.01).Low dose BBR intervention can significantly reduce the apoptosis of PVEc,which were presented as the increase of Bcl-2/Bax(p<0.01)and decrease of Caspase-3(p<0.05).Compared with the model group,the MD group and HD group significantly increased the expression of bcl-2 protein(p<0.05),but had no significant effect on Bax,bcl-2/Bax,and caspase-3.The LT group significantly reduced Bcl-2(p<0.01)and increased caspase-3(p<0.01).4.The ELISA results of NF-?B in pulmonary vascular tissue showed that:decompression injury significantly decreased the protein expression of NF-?B(p<0.01).Although the level of NF-?B after BBR intervention was still lower than that of the control group,there was a significant increase in LD and HD groups compared with model(p<0.05),and there was no significant difference to control.The changes of Bcl-2/Bax and Caspase-3 had a significant linear relationship with NF-?B(R~2=0.556 or0.486,p<0.01).5.The transcription level of apoptosis-related genes expression in pulmonary vascular tissues showed that:the transcription level of BaxmRNA was significantly increased after decompression injury(p<0.05),but there was no significant difference between model and control groups in Bcl-2mRNA and Caspase-3mRNA.It was showed lower transcription level of Bax gene in LD group than model(p<0.01).However,there were no differences between the MD or HD group.6.The results of vasoactive factors content in peripheral blood showed that NO contents of model group decreased significantly than that of control group(p<0.01).NO contents were increased significantly in BBR intervention LD,MD group and LT group(p<0.01 or p<0.05).NO of LT group was significantly higher than that of HD group(p<0.01).The NO content in peripheral blood was positively correlated with the dosage of BBR(r=0.773,p<0.01).7.Quantitative detection of NOS in lung tissue showed a significant decrease in NOS protein content(p<0.05)after decompression injury.Immuno-histochemical sections showed that eNOS was mainly distributed in vascular endothelium and alveolar epithelium with basic expression in control group.The coloration of model group decreased apparently.Results of three subtypes of NOS with WB showed that the three subtypes of NOS had a downward trend of protein expression in model but without significant difference.BBR pre-treatment increased NOS protein expression significantly in the MD group and the LT group(p<0.01 or p<0.05).There were no differences in NOS protein expression between the BBR intervention groups and the control group.The protein expression of eNOS of LT group was significantly higher than that in model group(p<0.01),and significantly higher than that in control group(p<0.05).The changes of iNOS were similar to that of eNOS,but not to the significant level.There was a significant predictive relationship between NOS content in lung tissue and NO content in peripheral blood(R~2=0.325,F=16.881,p<0.01),and the significant correlation between NOS content and dosage of BBR(r=0.730,p<0.01).8.The results of MAPK phosphorylation level in lung tissue showed that,the phosphorylation levels of JNK and p38 in model group were significantly decreased than control(p<0.05),while the phosphorylation levels of ERK were not significantly changes.The level of p-JNK in BBR intervention group was significantly higher than that in model group(p<0.01),and there was a correlation with dose(r=0.865,p<0.01).P-JNK in the LT group was significantly higher than that in the HD group(p<0.01).The changes of p-p38 after BBR intervention were significantly different from those of p-JNK(p<0.01or p<0.05).P-JNK and p-p38 in lung tissue were significantly correlated with NOS(R~2=0.609 or 0.552,p<0.01),and positively correlated with dosage(r=0.865 or0.856,p<0.01).Conclusion:1.Pathological changes,along with lung wet-dry weight ratio,DES and IL-1?results,suggested that decompression damages induced pulmonary interstitial edema,congestion,tissue destruction and inflammatory reaction through accumulation of intravascular bubbles in the lung.The results of apoptosis-related indicators of pulmonary vascular tissue suggested that decompression intravascular bubbles could lead to morphological and functional damage of pulmonary vascular endothelial cells,and even induced apoptosis.The results of NO in peripheral blood showed that decompression damages had effects on vasomotor function.Peripheral blood NO abnormality was correlated with changes of NOS protein expression in lung tissue,which suggested decompression damages'effects on vasoactive maybe more than vasoactive factor synthesis and secretion.There was a predictive relationship between NOS protein expression and MAPK phosphorylation level,which suggests that MAPK-NOS-NO pathway plays a role in decompression damages of lung tissue.These results confirmed that pulmonary vascular endothelium is an important target organ for decompression damages.The intravascular decompression bubbles can induce functional and genetic changes for synthesis and secretion of endothelium-derived relaxing factor by injuring pulmonary vascular endothelial cells.2.Our results confirmed BBR's effects in alleviation of decompression injury.The effects of BBR were supposed to effect through the following ways:(1)MAPK-NOS-NO pathway.BBR up-regulated phosphorylation levels of JNK and p38,but not ERK of MAPK,and then increased of eNOS mRNA transcription,increased the protein expression of eNOS in pulmonary vascular endothelium,which was followed with the increased synthesis and secretion of NO.The latter could achieve the ultimate goal of reducing decompression injury by relaxing blood vessels,inhibiting platelet aggregation and leukocyte activation;(2)NF-?B related mechanism.BBR up-regulated the protein expression of NF-?B in pulmonary vascular tissue after decompression stress,which was followed with down regulation of the Bax gene expression.And then,the apoptosis of PVEc induced by bubbles was inhibited to preserving vascular endothelial function and resisting decompression injury.3.BBR effects on MAPK-NOS-NO were dose-dependent.The phosphorylation level of JNK and p38,the NOS protein expression and the NO synthesis were changed company with the dose of BBR,which were conducive to maintaining reasonable pulmonary vasoactive function.However,high dose BBR may lead to excessive expansion of blood vasoactive factors,and along with the effects on NF-?B related mechanism of PVEs apoptosis.Clinical manifestations and other indicators of decompression stress also suggested that the comprehensive preventive effect of high dose BBR was not superior to that of medium and low dose.Therefore,in order to achieve the better effects of BBR pre-conditioning on DCS,it needs to make more efforts to explore ways to the keep balance between the effects of multiple targets with studies under the separate conditions of decompression stress.This study not only enrich the understanding of the mechanism of decompression sickness,but also discover the effect of single medication of TCM ingredient BBR pre-treatment on decompression stress by multi-pathway,which may highlight further exploring rapid drug intervention measures adapted to military needs. |
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