Regulation Of High Mobility Group Protein B1 Through Heme Oxygenase-1 Of Intervention In Lead Exposure

In this study,the changes of HMGB1 and HO-1 expression in lead exposed environment and the effects of HO-1 intervention on the expression of HMGB1 in lead exposed environment were studied.1.Effects of lead exposure on the expression of HMGB1 and HO-1 in hippocampus of weaning rats Objective:To investigate the expression of HMGB1 and HO-1 in the hippocampus of the weaning rats and the content of lead in hippocampus of weaning rats.Methods:(1)Animal modeling: the pregnant rats were randomly divided into 3 groups:(1)group S: control group,double distilled water;(2)group L: low lead exposure group,0.05%(0.5 g/L)lead acetate;(3)group H: high lead exposure group,0.2%(2.0 g/L)lead acetate.Lead exposure was performed using free drinking water until weaning(postnatal 21 days).(2)Experimental method:(1)Morris water maze was used to test the behavior of weaning rats;(2)The content of lead in whole blood and hippocampus of weaning rats was detected by ICP-AES;(3)The levels of HMGB1 and HO-1 mRNA in hippocampus of weanling rats was detected by RT-PCR;(4)The levels of HMGB1 and HO-1 protein in hippocampus of weaning rats was detected by WB;(5)The levels of HMGB1 and HO-1 protein in hippocampus were detected by IF.Results:(1)Mirros water maze experimental results: lead exposure will lead to weaning rats learning and memory capacity decreased.(1)The escape latency: Compared with C group,the latency of L group and H group was prolonged(P<0.05);(2)Through the platform number: compared with the C group,L group and H group significantly reduced the number of crossing platform(P<0.05)and the number of H group was lower than that of group L(2)Blood lead and hippocampus lead concentration test results: lead exposure can cause weaning rats blood lead and hippocampal lead levels rise.ICP-AES showed that lead concentration and hippocampal lead concentration in weanling rats were increased with the increase of lead exposure(P<0.05).(3)The effect of lead exposure on HMGB1 expression in weaning rats:(1)RT-PCR showed that the HMGB1 mRNA levels in hippocampus of weaning rats increased with the increase of lead exposure(P<0.05);(2)WB experiment showed that the levels of HMGB1 protein increased with the increase of lead exposure(P<0.05);(3)IF experiments showed that the levels of HMGB1 protein in hippocampus CA1,CA3 and DG increased with the increase of lead exposure(P<0.05).(4)The effect of lead exposure on the expression of HO-1 in weaning rats:(1)RT-PCR showed that the HO-1 m RNA levels in hippocampus of weaning rats was increased with the increase of lead exposure(P<0.05);(2)WB experiment showed that the levels of HO-1 protein in the weaning rats was higher than that in the C group with the increase of lead exposure concentration(P<0.05);(3)IF experiments showed that the levels of HO-1 protein in the hippocampus CA1,CA3 and DG region was higher than that in the C group(P<0.05).2.Effects of lead exposure on the expression of HMGB1 and HO-1 in PC12 cells Objective:The PC12 cell lead exposure model was established to observe the expression of HMGB1 and HO-1.Methods:(1)Cell modeling: PC12 cells were divided into 3 groups: 0 ?M(control group),1?M and 100?M lead acetate.lead treatment for 24 h.(2)Experimental method:(1)The levels of HMGB1 and HO-1 m RNA in PC12 cells was detected by RT-PCR;(2)The levels of HMGB1 and HO-1 protein in PC12 cells was detected by WB;(3)The levels of HMGB1 and HO-1 in PC12 cells was detected at different time(24h,48 h,72h)by IF.Results:(1)The effect of lead exposure on the expression of HMGB1 in PC12 cells:(1)RT-PCR experiments showed that the levels of HMGB1 mRNA was increased by lead acetate in a concentration-dependent manner(P<0.05);(2)WB experiment showed that the levels of HMGB1 protein was increased by lead acetate in a concentration-dependent manner(P<0.05);(3)IF experiments showed that the levels of HMGB1 protein in PC12 cells was increased at different time(24h,48 h,72h)by lead acetate in a concentration-dependent manner(P<0.05).(2)The effect of lead exposure on the expression of HO-1 in PC12 cells:(1)RT-PCR experiments showed that the levels of HO-1 mRNA was increased by lead acetate in a concentration-dependent manner(P<0.05);(2)WB experiment showed that the levels of HO-1 protein was increased by lead acetate in a concentration-dependent manner(P<0.05);(3)IF experiments showed that the levels of HO-1 protein in PC12 cells was increased at different time(24h,48 h,72h)by lead acetate in a concentration-dependent manner(P<0.05).3.Effect of HO-1 intervention on the expression of HMGB1Objective:The expression of HMGB1 was observed by adding HO-1 inhibitor ZnPP-? or agonist CoPP to the lead exposure cell model.Methods:(1)Cell modeling: PC12 cells were divided into 4 groups:(1)group S: control group,0 ?M PbAc;(2)group PA : PbAc(1 ?M);(3)group Zn: PbAc(1 ?M)+ ZnPP(20 ?M)and(4)group Co: PbAc(1 ?M)+ CoPP(20 ?M).Treatment of 12 h with lead exposure followed by addition of an interfering agent to 12 h.(2)Experimental method:(1)The mRNA levels of HMGB1 and HO-1 in PC12 cells was detected by RT-PCR;(2)The protein levels of HMGB1 and HO-1 in PC12 cells was detected by WB.Results:(1)(1)Compared with the S group,the levels of HO-1 mRNA and protein in PA group and Co group were increased(P<0.05);(2)Compared with PA group,the expression of HO-1 in Zn group was decreased(P<0.05),in Co group was increased(P<0.05).(2)(1)Compared with the S group,the levels of HMGB1 mRNA and protein in PA group and Zn group were higher(P<0.05).(2)Compared with PA group,The expression of HMGB1 in Zn group was increased(P<0.05),in Co group was decreased(P<0.05).Conclusion:1.Lead exposure increases the expression of HO-1 and HMGB1.2.The up-regulation of HO-1 in lead exposure environment inhibited the expression of HMGB1.