Effect Of Vitamin D On The Expression Of Local Renin Angiotensin System In Rats With Acute Lung Injury Induced By Endotoxin

BackgroundAcute lung injury?ALI?and acute respiratory distress syndrome?ARDS?is a clinical critical disease which serious threat to human health and life,has aroused great attention for scholars both foreign and chinas,but its pathogenesis has not been clarified.Vitamin D?Vit D?participates in many processes such as immune response,cell growth and apoptosis through its receptor in the target nucleus.The renin-angiotensin system?RAS?is widely involved in the regulation of body function,inflammation,immunity,apoptosis,growth,aging,metabolism,tissue repair,reproductive development,nerve conduction,learning,memory and other physiological activities.By changing the release of a variety of inflammatory cells and inflammatory factors,RAS leads to an imbalance of systemic inflammatory response and imbalance of proinflammatory and anti-inflammatory mechanisms in the lung,thus affecting the development and development of lung injury.A number of studies have shown that the ACE-Ang II-AT1axis contributes to pulmonary fibrosis,while the ACE2-Ang?1-7?-Mas axis inhibits it.Studies have shown that Vit D can inhibit the pathophysiology ofRAS mediated by local and cellular processes.At present,a lot of studies are about kidney disease,diabetes mellitus,cardiovascular disease and so on.But there are few reports in the lung tissues.The specific pathophysiology of Vit D mediated RAS mediated ALI remains to be elaborated.We predict that in ALI,Vit D play a negative regulatory role on the occurrence and development of ALI,by inhibiting the expression of ACE-Ang II-AT₁axis and improving the expression of ACE2.Further elucidate the effects of Vit D on the expression of RAS in the pathogenesis of ALI/ARDS,further enrich the pathophysiological basis of ALI pathogenesis,and provide a new idea for the prevention and treatment of ALI.ObjectiveTo research the expression change of renin angiotensin system?RAS?members in the process of LPS induced acute lung injury?ALI?in lung tissue of rats and pulmonary micro vascular endothelial cells?PMVEC?,and the influence of vitamin D?vit D?on the expression changes.To discuss the changes of the RAS members expression,and the effect rule of Vit D on the expression of REN,ANG,ACE,ACE2,AT₁R and AT₂R in lung tissue in the process of ALI induced by LPS.To clarify the effect of Vit D on RAS members in the pathogenesis of ALI,to further enrich the pathophysiological basis of ALI,and to provide a new idea for the prevention and treatment of ALI.MethodsIn vivo experiment:the rats were randomly divided into 6 groups.Normal control?NC?group:the equivalent amount of normal saline was injected into the tail vein,and observation 24 hours;LPS group:LPS?5mg/kg?tail vein injection,and observation 24hours;calcitriol?Cal?group:the rats were treated with calcitriol?25ug/kg.d?for three days;LPS+Cal 1 groups:the rats were treated with calcitriol?1ug/kg.d?for three days,and injected the LPS?5mg/kg?by tail vein at the third day,then observation 24 hours;LPS+Cal 2 groups:the rats were treated with calcitriol?5ug/kg.d?for three days,and injected the LPS?5mg/kg?by tail vein at the third day,then observation 24 hours;LPS+Cal 3 groups:the rats were treated with calcitriol?25ug/kg.d?for three days,and injected the LPS?5mg/kg?by tail vein at the third day,then observation 24 hours.Observate the lung tissue pathology,Bronchoalveolar lavage fluid cell count,lung wet/dry weight ratio,and Evans blue penetration in each group rats,qReal time PCR was used to detect lung tissue renin,Ang II,ACE,ACE2,AT₁R and AT₂R mRNA expression changes,Western blot was used to detect lung tissue ACE,ACE2,AT₁R and AT₂R protein expression changes,ELISA was used to detecte renin and Ang II protein expression changes.In vitro experiment:Rat pulmonary microvascular endothelial cells?PMVEC?were isolated and cultured,and then randomly divided into 6 groups.NC group:PMVECs were added 3ml DMEM which contain 1%FCS,then incubated with 24h;LPS group:PMVECs were added 3ml DMEM which contain 1%FCS and 100 ug/ml LPS,then incubated with 24h;Cal group:PMVECs were added 3ml DMEM which contain 1%FCS and 100nmol/l calcitriol,then incubated with 24h;LPS+Cal 1 group:PMVECs were added 3ml DMEM which contain 1%FCS,5nmol/l calcitriol and 100 ug/ml LPS,then incubated with 24h;LPS+Cal 2 group:PMVECs were added 3ml DMEM which contain 1%FCS,20nmol/l calcitriol and 100 ug/ml LPS,then incubated with 24h;LPS+Cal 3 group:PMVECs were added 3ml DMEM which contain 1%FCS,100nmol/l calcitriol and 100 ug/ml LPS,then incubated with 24h.qReal time PCR was used to detect renin,Ang II,ACE,ACE2,AT₁R and AT₂R mRNA expression changes in PMVECs,Western blot was used to detect ACE,ACE2,AT₁R and AT₂R protein expression changes in PMVECs,ELISA was used to detecte renin and Ang II protein expression changes in PMVECs.ResultsIn vivo experiment,we have successfully replicated acute lung injury rat model induced by LPS.Cell counts of bronchoalveolar lavage fluid were significantly increased in LPS group and each LPS+Cal group compared with NC group,LPS+Cal 2group and LPS+Cal 3 group were significantly lower than that of LPS group.Results of lung wet/dry weight ratio,LPS group and each LPS+Cal group were significantly higher than those in NC group,each LPS+Cal group significantly decreased compared with LPS group.Evans blue bleeding results,LPS group and each LPS+Cal group were significantly reduced compared with NC group,LPS+Cal 2 group and the LPS+Cal 3group was significantly reduced compared with LPS group.QRT-PCR test results:In ACE mRNA expression,LPS group and each LPS+Cal group was significantly higher than the NC group,and LPS+Cal 3 group was significantly lower than the LPS group.The expression of mRNA ACE2 in LPS group and LPS+Cal1 group was significantly lower than that in NC group.In AT1R mRNA expression,LPS group and each LPS+Cal group was significantly increased than NC group,and LPS+Cal2 group and LPS+Cal2 group was significantly lower than the LPS group.There was no significant difference in the expression of AT2R mRNA in each group.The expression of REN mRNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of REN mRNA in LPS+Cal 2 group and 3 group was significantly lower than that in LPS group.The expression of Ang II mRNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of Ang II mRNA in LPS+Cal 2 group and LPS+Cal 3 group was significantly lower than that in LPS group.Blot Western detection results:The expression of ACE protein in LPS group,LPS+Cal 1 group and LPS+Cal 2 group was significantly higher than that in NC group,the expression of ACE protein in LPS+Cal 3 group was significantly lower than that in LPS group.The expression of ACE2 protein in LPS group,LPS+Cal 1 group and 2group was significantly lower than that in NC group,the expression of ACE2 protein in LPS+Cal 3 group was significantly higher than that in LPS group.The expression of AT1R protein in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of AT1R protein in LPS+Cal 3 group was significantly lower than that in LPS group.There was no significant difference in the expression of AT2R protein in each group.ELISA results:The expression of REN protein in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of REN protein in LPS+Cal 2 group and 3 group was significantly lower than that in LPS group.The expression of Ang?protein in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of Ang?protein in each LPS+Cal group was significantly lower than that in LPS group.In vitro experiment part:The primary rat PMVEC was successfully cultured and subculture.QRT-PCR test results,the expression of ACE mRNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of ACE mRNA in LPS+Cal3 group was significantly lower than that in LPS group;the expression of ACE2 mRNA in LPS group and each LPS+Cal group was significantly lower than that in NC group,the expression of ACE2 mRNA in each LPS+Cal group was significantly higher than that in LPS group;the expression of AT1R m RNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of AT1R mRNA in LPS+Cal3 group was significantly lower than that in LPS group;there was no significant difference in the expression of AT2R mRNA in the each group;the expression of REN mRNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of REN mRNA in LPS+Cal 2group and 3 group was significantly lower than that in LPS group;the expression of Ang II mRNA in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of Ang II mRNA in each LPS+Cal group was significantly lower than that in LPS group.Western-blot detection results,LPS group,each LPS+Cal group in ACE protein expression was significantly increased compared with NC group,LPS+Cal2 group and 3 group in ACE protein expression was significantly lower than that of LPS group;the expression of ACE2 protein in LPS group and each LPS+Cal group was significantly lower than that in NC group,the expression of ACE2 protein in LPS+Cal 2 group and 3 group was significantly higher than that in LPS group;the expression of AT₁R protein in LPS group and each LPS+Cal group was significantly lower than that in NC group,the expression of AT₁R protein in LPS+Cal 3 group was significantly lower than that in LPS group;there was no significant difference in AT₂R protein expression between the groups.ELISA detection results,the expression of REN protein in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of REN protein in LPS+Cal 2 group and 3 group was significantly lower than that in LPS group;the expression of Ang II protein in LPS group and each LPS+Cal group was significantly higher than that in NC group,the expression of Ang II protein in LPS+Cal 3 group was significantly lower than that in LPS group.Conclusions1.LPS up-regulated the expression of REN,An gII,ACE,AT₁R gene and protein and down-regulated the expression of ACE2 gene and protein in rat lung tissue and rat PMVEC.2.Vit D can inhibit the aforementioned role of LPS.3.AT₂R had no significant changes in local expression of lung during the development of ALI,and Vit D has no effect on its expression.4.In the pathogenesis of ALI,Vit D involved in the occurrence and development of the disease through the different effects on the expression of the members of the local RAS.