Study On The Protection Effect And Mechanisms For Paeoniflorin Preventing Myocardial Dysfunction Via A1 Adenosine Receptor In Septic Mices

Part I The effects of paeoniflorin on the mortality of septic micesObjective To investigate the appropriate Pae dosage and research its mechanism and role in septic mices by counting their mortality.Method 1.1 To observe the effects of different dosage of paeoniflorin on the mortality of septic mices.Establishing CLP models with sixty SPF Kunming male mice which weight from 30 g to 35 g.These mice were randomly divided into six groups.①Sham group,deal with distilled water. ② CLP+ distilled water. ③ CLP+ Pae(10mg/kg). ④ CLP+ Pae(20mg/kg).⑤CLP+ Pae(40mg/kg).⑥CLP+ Pae(80mg/kg).In the Sham group and the control group, mices were respectively given distilled water in a dose of 0.1ml/10 g and above different dose of paeoniflorin,4 hours and continuous 3 days after the CLP operation.We choose the appropriate dosage by counting the mortality of each group with in 10 days.1.2 The effects of different dosage of DPCPX on the mortality of septic mices.The mices were divided into five groups: ① CLP+NS. ② CLP+DMSO. ③DPCPX(1mg/kg)+CLP.④CLP+DPCPX(2mg/kg).⑤CLP+DPCPX(4mg/kg).The first two groups were given a subcutaneous injection of NS or DMSO in a dose of 0.1ml/10 g,30 minutes before and 3.5 hours after the CLP operation.Other groups were given a subcutaneous injection of different does of DPCPX.Observing the mortality of each group in 10 days.,we screening the appropriate DPCPX dosage in our experimental conditions.1.3 To observe the effect of paeoniflorin on the mortality of septic mices after using DPCPX.The mices were divided into four groups:①CLP+ DMSO+H2O.②CLP+ DMSO+Pae.③CLP+ DPCPX+ H2 O.④CLP+ DPCPX+Pae. All groups were given a subcutaneous injection of DPCPX(1mg/kg) or DMSO(0.1ml/10g), 30 minutes before and 3.5 hours and continuous 3 days after the CLP operation. All groups were given a lavage injection of Pae(20mg/kg) or distilled water(0.1ml/10g), 4 hours and continuous 3 days after their surgery. Observing the mortality of each group in 10 days.Result 1.The septic mices’ mortality is 10 percent at a dose of 20mg/kg, its average survival time is(9.1±2.8) days, longer than other CLP groups(P<0.05). It shows that dose can remarkably improve septic symptoms,so that reducing the mortality.2.DPCPX(1mg/kg) groups’ mortality is 55 percent. And its average survival time is(5.7±1.2) days, longer than the dose of 4mg/kg(2.6±0.67),but no difference with the dose of 2mg/kg(P>0.05).3.The survival rates in 10 days in CLP+DPCPX + H2 O group are 90.9%, after using DPCPX.Its average lifetime is 9.182±0.78 days, significantly longer than CLP+DMSO+H2O groups(6.7±1.1d)(P<0.05).And the CLP+DMSO +Pae group’s average lifetime is longer than CLP+DPCPX +Pae group(P<0.05).So DPCPX could aggravate the rat’s septic symtoms and inhibit the protective effect of Pae on sepsis in mice.Conclusion The Pae is very effective to the protection of the mices’ septic symtoms at a dose of 20mg/kg.The Pae is play a role in the treatment of sepsis via adenosine receptors.Part II Pae affect the function of septic mices after using adenosine receptor antagonist.Objective Based on mortality, research Pae protecting organ function in septic mice.Method Experimental models were designed as follows. ①CLP+ DMSO+ H2 O.②CLP+ DMSO +Pae.③CLP+ DPCPX+ H2 O. ④ CLP+ DPCPX+Pae. ⑤ Sham+ DMSO+ H2 O. ⑥ Sham+ DMSO+ Pae; ⑦ Sham+ DPCPX+ H2 O. ⑧ Sham + DPCPX+Pae. Sham groups and CLP groups have the same way and time to give Pae and DPCPX. Serum samples were collected to detect AST,ALT,BUN,CR,12 hours and 24 hours later.Result 1. At time of 12 h, the level of ALT,AST CR,BUN in the CLP groups was obviously higher than the Sham groups(P<0.05).Similarly the level of CR,BUN, the CLP+ DMSO +Pae group was higher than the CLP+ DMSO+ H2 O group(P<0.05). And the level of AST of the CLP+DPCPX + H2 Ogroup was high than the CLP+DMSO +H2O group(P<0.05).2.There is no difference of statistics in each groups on the level of CR,BUN for 24 hours after the CLP surgery. On the level of AST, the CLP+DMSO +H2O group is higher than the CLP+DMSO +Pae group.,but lower than the CLP+ H2O+DPCPX group on the level of ALT,AST.Conclusion Pae is able to reduce septic symptoms and mitigate the damage of organs, but it can be blocked by DPCPX.Part III To explore Pae pretect the septic mices’ myocardial dysfunction through A1 R.Objective To explore Pae pretect the septic mices’ myocardial dysfunction.Method This part of experiment has the same grouping and processing with part II. We tevaluate HR,SV,CO etc by animal ultrasound system,12 hs and 24 hs after the CLP operation.. Take the myocardial tissue to measure the levels of LDH activity,24 hours after the CLP operation.Result All CLP groups for 12 hours and 24 hours has the same result that SV,CO,E/A has decreased and a systolic and diastolic myocardial dysfunction. Among of them,the CLP+DMSO +Pae group is relatively higer than other group(P<0.05).Results about LDH: The CLP+DMSO +H2O group’s LDH activities are remarkably higher than the Sham+DMSO +H2O group( P<0.05), but lower than the CLP+DPCPX +H2O group.And the CLP+DMSO +Pae group’s LDH activities are obviously lower than the others.Conclusion Pae plays a role in improving the myocardial contractile and diastolic function by activing the A1 adenosine receptor.Part Ⅳ To explore the mechanism of Pae pretecting the septic mices’myocardial dysfunction through A1 R.Objective To explore the mechanism of Pae pretecting the septic mices’ myocardial dysfunction via A1 R..Method This part of experiment has the same grouping and processing as former.Take the myocardial tissue to measure the levels of SOD activity and MDA content 24 hours after the CLP operation.Result CLP+DMSO + H2 O group has a lower activity of SOD than Sham group(P < 0.05). CLP+DMSO +Pae group has a higher activity of SOD than other model groups(P < 0.05). CLP+DMSO + H2 O has a higher content of MDA than Sham group(P < 0.05). CLP+DMSO +Pae group has has a lower content of MDA than other model groups(P < 0.05).Conclusion Pae has a strong influence on oxygen free radicals metabolism in myocardial tissue, increasing the level of SOD activity, reducing lipid peroxidation damage in myocardial tissue.