The Role Of MT2 On LPS Tolerance Of Mice

Objectives MT2 gene knockout homozygous(MT2-/-)mice were obtained by CRISPR technology.The endotoxin tolerance models were constructed using MT2-/-mice and wild-type mice,respectively.Our aim was to observe the differences in serum inflammatory cytokines,liver pathological damage,and the phosphorylation of p38 MAPK in mouse liver of different genotypes.Effects of metallothionein 2 on endotoxin tolerance and its mechanism in this study may provide new ideas for MT2 and more advisable treatment for the sepsis.Methods1.Acquisition of MT2-/-mice The heterozygous MT2 +/-mice were obtained by CRISPR / Cas9 technique.MT2+/-mice were hybridized and backcrossed to obtain progeny.Progeny were identified by PCR,WB detection and gene sequencing technology.The MT2-/-mice and wild-type mice were used for the experiment.2.Endotoxin tolerance test Different genotypes of mice were randomly divided into six groups:wild type control group(WT Ctrl group),wild type high dose LPS stimulation group(WT LPS group),wild type tolerance group(WT ET group),MT2-/-control group(MT2-/-Ctrl group),MT2-/-high dose LPS group(MT2-/-LPS group),MT2-/-endotoxin tolerance group(MT2-/-ET group).The survival status and survival number of each group were observed and analyzed.3.Observation of endotoxin tolerance effect We repeat the endotoxin tolerance approach.At the time of 12 h,1d,2d,3d,4d and 5d after stimulation of high dose LPS,three mice were sacrificed in each group.Expression of TNF-??IL-10?ALT in serum and expression of MDA?SOD in liver homogenate were detected by ELISA.Morphologic changes of mice's liver were examined by HE staining,the apoptosis index of liver were detected by TUNEL.Western blot and Immunohistochemistry were employed to observe levels of phosphorylated p38 MAPK in mice's liver attcked by LPS.Results(1)We obtained a sufficient number of MT2 knocker mice for later experimental studies(2)The model of endotoxin tolerance was successfully constructed in MT2 knockout mice and wild-type mice.MT2-/-ET required 5 days to recover from endotoxin attack,while WT ET required 3 days.After the high dose endotoxin attck,the death time of MT2-/-LPS mice was concentrated within 36 h,while the WT LPS group was concentrated within 48 hours.Compared with the same time node,the mortality rate of WT LPS group was more high(P <0.05).(3)The levels of proinflammatory cytokine TNF-? and ALT in MT2-/-ET group are higher than those of WT ET group(P<0.05),and the peak time is shorter.The level of anti-inflammatory factor IL-10 was lower than that of WT ET group(P<0.001).(4)The level of MDA in MT2-/-ET was significantly higher than that of the control group(P<0.001),the activity of SOD was significantly decreased(P<0.001).The level of MDA in ET group was lower than that in MT2-/-ET(P<0.05).(5)The expression of p-p38 MAPK protein was lower in MT2-/-ET group compared with WT ET group.Conclusion MT2-/-mice were successfully obtained and used to construct endotoxin tolerance model.MT2-/-mice take longer to recover after endotoxin strikes.MT2 could inhibit the release of TNF-? after endotoxin stimulation,which promote the release of anti-inflammatory factor IL-10.Moreover,MT2 may reduce liver pathological damage by the inhibition of p-p38 protein expression in liver.The anti-inflammatory effect of MT2 may be related to the inhibition of p38 MAPK signaling pathway.