| Objective:The rat model of endotoxemia was reproduced by intraperitoneal injection of bacterial lipopolysaccharide 10mg/kg.To investigate the effect of triptolide?TP?on cardiovascular function in rats with endotoxemia.At the same time,HUVEC cells were co-cultured with LPS to observe the effect of TP on LPS-stimulated inflammatory damage in HUVEC cells.The aim of this study was to explore the new target and basis of TP in the prevention and treatment of endotoxemia.Method:1.Animal experimentSixty male SD rats were injected intraperitoneally with 10 mg/kg Lipopolysaccharide?L82749,LPS,026:B6?to replicate the model of endotoxemia.TP intervention group was pretreated 15 minutes before LPS was injected intraperitoneally.Rats were divided into normal group?NC group?,endotoxemia model group?LPS group?,low TP concentration intervention group?LPS+TP-L group,25?g/kg?,medium TP concentration intervention group?LPS+TP-M group,50?g/kg?,high TP concentration intervention group?LPS+TP-H group,100?g/kg?and polymyxin B group?LPS+PMX-B group,0.2 mg/kg?.Common carotid artery intubation in rats of each group.First,the mean arterial blood pressure?MABP?were measured in each group.Then the hemodynamic parameters were measured,including heart rate?HR?,left ventricular diastolic pressure?LVDP?,maximum rate of increase/decrease of left ventricular pressure,+dp/dtmax.The levels of BNP,CK-MB and cTn-I in serum and TNF-?and IL-6 in plasma were detected by ELISA.Western Blot method was used to detect the content of p65 protein in myocardium and iNOS,eNOS,TLR4 and p65 protein in thoracic aorta.2.Vascular ring tension test:The grouping of in vitro and in vivo experimental parts is the same.After hemodynamic parameters were measured,thoracic aortic rings were taken from rats in each group.Changes of ring tension were recorded by PL3508B5/C-V tissue perfusion device in vitro and Power Lab system.3.HUVEC cell experiment:Normal cultured HUVEC cells were randomly divided into normal control group?NC group?,LPS group and TP+LPS group.NC group:cells were cultured normally without any other treatment;LPS group:cells were stimulated with LPS of 1000 ng/ml for 12 hours on the basis of normal culture;TP+LPS group:TP?concentration of 0,0.25,0.5,1,2,4,8,10,20,30,50,100,200,300,400,500,800,1000?g/ml?was screened out by CCK-8 method for the Safe range of HUVEC cells,and Safe concentration of TP(concentration of TP was0,0.25,0.5,1,4,8,20,30,100,200,300,HUVEC cells were pretreated for 2 hours,then co-cultured with LPS of 1000 ng/ml.The supernatants of HUVEC cells were collected for0,1,2,3,4,5,6,8,12,16,18,20,24,30,36 and 48 hours respectively.The supernatants of NC and LPS groups were collected only for 12 hours.The changes of TNF-?and IL-6contents in the supernatants of each group were detected by ELISA.Cell grouping is the same as above.Cell proteins were extracted for 0,6,12,18 and 24hours in the TP+LPS group respectively.Cell proteins were extracted for 12 hours only in NC and LPS groups.The changes of eNOS,TLR4 and p65 contents in each group were detected by Western Blot method.Result:1.Animal experiments?1?Changes of hemodynamics in rats of each groupCompared with NC group,the MABP and the hemodynamic indexes such as HR,LVDP,+dp/dtmax in LPS group decreased significantly?P<0.05?,while MABP,HR,LVDP,+dp/dtmax in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group did not change significantly?P>0.05?.Compared with LPS group,the MABP of rats in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group was significantly improved?P<0.05?;the HR,+dp/dtmax of rats in LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group were significantly improved?P<0.05?,and the LVDP of rats in LPS+TP-H group and LPS+PMX-B group recovered significantly?P<0.05?.?2?Contents of BNP,CK-MB and cTn-I in serum of rats in each groupCompared with NC group,serum BNP levels in LPS group,LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group increased significantly?P<0.05?;serum CK-MB and cTn-I levels in LPS group,LPS+TP-L group and LPS+TP-M group increased significantly?P<0.05?,while there was no significant change in LPS+TP-H group and LPS+PMX-B group?P>0.05?.Compared with LPS group,the contents of BNP in LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?;the contents of CK-MB and cTn-I in serum of LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?;the contents of CK-MB and cTn-I in serum of LPS+TP-H group and LPS+PMX-B group did not change significantly?P>0.05?.?3?The contents of TNF-?and IL-6 in plasma of rats in each groupCompared with NC group,TNF-?and IL-6 in LPS group,LPS+TP-L group and LPS+TP-M group increased significantly?P<0.05?,while TNF-?and IL-6 in LPS+TP-H group and LPS+PMX-B group did not change significantly?P>0.05?.Compared with LPS group,the contents of TNF-?and IL-6 in LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?.?4?The content of p65 in myocardial tissue,iNOS,eNOS,TLR4 and p65 in vascular tissue of rats in each groupCompared with NC group,the contents of p65 in LPS group,LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and myocardial tissue increased significantly?P<0.05?;compared with LPS group,the contents of p65 in myocardial tissue of LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?;compared with LPS+TP-L group,the contents of p65 in myocardial tissue of LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?.Compared with NC group,the contents of eNOS,TLR4 and p65 in LPS group,LPS+TP-L group,LPS+TP-M group,LPS+TP-H group,LPS+PMX-B group increased significantly?P<0.05?;the contents of iNOS in LPS group,LPS+TP-L group and LPS+TP-M group increased significantly?P<0.05?;the contents of iNOS in LPS+TP-H group and LPS+TP-B group had no significant difference?P>0.05?.Compared with LPS group,the contents of iNOS and eNOS in LPS+TP-L group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?;the contents of TLR4 and p65 in LPS+TP-L group,LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?.Compared with LPS+TP-L group,the contents of iNOS and p65 in LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?.The contents of eNOS and TLR4 in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group decreased significantly?P<0.05?.2.Changes in the tension of the thoracic aorta rings in each group?1?Changes in TP-induced contraction of thoracic aorta rings?E+,E-?induced by PheCompared with the NC group,the Phe of 10-810-5mol/L significantly reduced the contraction rate of the thoracic aorta ring?E+?in the LPS group?P<0.05?;and the contraction rate of the thoracic aorta rings?E-?in the LPS group was significantly decreased?P<0.05?.Compared with the LPS group,the contraction rate of the thoracic aorta ring?E+?in the TP intervention group and the LPS+PMX-B group increased significantly and increased in a concentration-dependent manner?P<0.05?;there was no significant change in the contraction rate of the thoracic aorta rings?E-?of the 10-810-5 mol/L Phe-stimulated endothelium in the TP intervention group?P>0.05?.?2?Effect of TP on ACh-induced thoracic aortic ring?E+?diastolic responseCompared with the NC group,the relaxation rate of the thoracic aorta ring?E+?in the LPS group was significantly decreased?P<0.05?at 10-810-5mol/L ACh stimulation and the diastolic rate of thoracic aortic rings in TP intervention groups increased in a concentration-dependent manner.Compared with LPS group,the diastolic rate of thoracic aortic rings?E+?induced by 10-810-5mol/L ACh in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group increased significantly?P>0.05?.?3?Effect of TP on Phe-induced L-NAME and MB preconditioning of thoracic aorta rings?E+?The thoracic aortic rings were pretreated with 10-4 mol/L L-NAME or 10-55 mol/L MB for 20 min,respectively.The thoracic aorta rings?E+?in each group were 10-810-5 mol/L Phe.Under the stimulation,the contraction amplitude increased.Before and after pretreatment with 10-4mol/L L-NAME,the difference in contraction rate between the LPS group's thoracic aorta ring?E+?and 10-5mol/L Phe was?61.55±14.54 vs 43.09±9.75,P=0.023?;?61.55±14.54 vs 36.75±17.98,P=0.018?;significantly higher than LPS+TP-H group and NC group.Before and after 10-5 mol/L MB pretreatment,the difference in contraction rate between the LPS group and the thoracic aorta ring?E+?to 10-55 mol/L Phe was?49.59±11.49 vs 32.27±3.87,P=0.023?.?49.59±11.49 vs 29.74±11.39,P=0.044?;significantly higher than the LPS+TP-H group and the NC group.3.LPS co-cultured HUVEC cells experimental part?1?CCK-8 screens the safe range of TP on HUVEC cellsCCK-8 screened out that the safe range of TP to HUVEC was 030?g/ml.The maximum concentration of 30?g/ml was selected as the concentration of the subsequent experiment.?2?Changes of TNF-?and IL-6 levels in HUVEC cells of each groupThe ELISA results showed that the levels of TNF-?and IL-6 in HUVEC cells of LPS group were significantly higher than those in NC group?P<0.05?.Compared with LPS group,TP+LPS group could decrease HUVEC cells at different time points.The levels of TNF-?and IL-6 in the group were significantly higher than those in the 0h group?P<0.05?,5h,6h,The levels of IL-6 in the cells at 20h,24h and 30h were significantly increased?P<0.05?.?3?Expression of TLR4,p65 and eNOS proteins in HUVEC cells of each groupThe expressions of TLR4,p65 and eNOS protein were detected by Western Blot.Compared with NC group,the expression of eNOS protein in HUVEC cells was significantly increased in LPS,0h,6h,12h,18h and 24h groups?P<0.05?;the expression of TLR4 protein in HUVEC cells was significantly increased in the 0h,6h,12h,18h and 24h groups?P<0.05?.The expression of p65 protein in HUVEC cells was significantly increased in LPS,0h,6h groups and the expression was significantly decreased in 24h group?P<0.05?.Compared with LPS group,the expression of eNOS protein in HUVEC cells was significantly increased at 0h,6h,12h,18h and 24h groups?P<0.05?.The expression of TLR4 protein in HUVEC cells was significantly decreased at 0h,6h,12h,18h and 24h groups?P<0.05?.The expression of p65 protein was significantly decreased in HUVEC cells at 0h,12h,18h and24h groups?P<0.05?.Compared with the 0h group,the expression of eNOS protein in HUVEC cells was significantly increased in the 6h and 18h groups?P<0.05?.The expression of p65 protein in HUVEC cells was significantly decreased in the 12h,18h and 24h groups?P<0.05?.The results showed that the acute injury of HUVEC cells induced by LPS could be alleviated in 12h,18h and 24h groups.Conclusion:1.TP has a protective effect on cardiovascular damage in endotoxemia rats in a dose-dependent manner;2.TP improves vascular hyporesponsiveness in endotoxemia rats and is associated with improved endothelial function.3.LPS co-cultured HUVEC cells can cause acute inflammatory damage,safe dose of TP?30 ug/ml?can alleviate LPS-induced HUVEC cell damage;TP may play an anti-inflammatory role in LPS-stimulated vascular endothelial injury through TLR4/NF-kappa Bp65 signaling pathway. |
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