The Effect Of Lipid Bound ApoA-I Cysteine Mutants On Lipopolysaccharide Induced Endotoxemia In Mice

High density lipoprotein(HDL) has been shown able to neutralize the toxicity of the lipopolysaccharide(LPS).ApoA-I is the principal protein constituent in HDL,and it is the key factor in the neutralization of the lipopolysaccharide.According to the mutant site of two natural mutants:ApoA-Imilano,ApoA-Ipairs and Edmunson wheels,our lab have succeeded in reconstituting seven ApoA-I cysteine mutants including ApoA-Imilano(A-I(S52C),A-I(N74C),A-I(K107C),A-I(G129C),A-I_M(R173C),A-I(K195C),A-I(S228C)),and the results showed that these mutants had different structural features or biological activities,suggesting the potential influence of the in vivo function of these mutations.AIM:In this study,we compared the anti-inflammation properties of wild-type apoA-I (wtapoA-I) and its 7 cysteine substitution mutants in lipid bound state.Our aim is to explore how the replacement of cysteine influences the structure and function of apoA-I,and whether there is a relationship between the structures and functions of mutants with specific cysteine mutant sites.We hope that our findings could shed some lights on the anti-inflammation mechanisms of HDL.METHODS:Both of wtapoA-I and the cysteine mutants were expressed with pET30b(+) as the expression vector and BL21(DE3) as the host bacterial,respectively.After purified by Ni²⁺ affinity chromatography,all of the proteins treated by Triton X-114 and Detoxi-GelTM Endotoxin Removing Gel in order to remove any residual endotoxin.Recombinant HDLs were prepared by using sodium cholate dialysis technique,then their in vivo anti-inflammation functions were examined by the septic mice which was induced by LPS.In order to assess the anti-inflammation of these rHDLs perfectly,we choose 3h and 24h two time point to measure the level of pro-inflammation cytokines in plasma by ELISA kits.Twenty four hours after LPS injection,lung was isolated from mice and fixed in 10%formaldehyde solution at room temperature,sectioned followed by stained with hematoxylin-eosin(H&E).In order to further explore the anti-inflammation mechanism of rHDL,we examine the ratios of DPPC to apoA-I in these rHDLs.RESLUTS:The construction of endotoxemia mice modelLPS was injected into mice through tail vein.After the anal temperature increased 0.5℃after LPS injection,we can get the endotoxemia model.Detection the level of inflammatory cytokines in the plasma of the mice24h post LPS injection,compared to controls injected with rHDLwt (135.28±12.84pg/ml),mice receiving either rHDL74 or rHDL52 exhibited significantly lower plasma levels of TNF-α(rHDL74:24.47±3.96pg/ml, P=0.002＜0.05 vs.rHDLwt;rHDL52:39.96±2.44pg/ml,P=0.009＜0.05 vs.rHDLwt). The plasma concentration of TNF-αin rHDL74 treated mice was reduced down to the baseline level(24.16±1.63pg/ml).Differently,mice treated with rHDL195 or rHDL228 had much higher plasma concentration of TNF-αcompared with LPS single injection groups(rHDL195:P=0.037＜0.05,rHDL228:P＜0.001,respectively). Treatment with recombinant HDL containing wt apoA-I also resulted in a significant decrease of plasma TNF-α(135.28±12.84pg/ml,P=0.045＜0.05 vs.LPS group). However,no statistical differences were observed in other groups of mice which were treated with rHDL107,rHDL129 or rHDL173 compared with LPS group.We also observed that the treatment of rHDLwt and all the other rHDL mutants except rHDL228 resulted in a decrease of plasma IL-1β(P＜0.001 vs.LPS).Mice treated with rHDL74 and rHDL52 had significantly lower secretion of IL-1βthan those treated with rHDLwt,(rHDL74:P＜0.001;rHDL52:P=0.021＜0.05,respectively). And other rHDLs(rHDL107,rHDL129,rHDL173 or rHDL195,P＜0.001,compared with LPS) also showed the capability of decreasing plasma IL-1βinduced by LPS injection.Besides these,we also found that treating mice with rHDL74 significantly attenuated plasma IL-6 production induced by in vivo LPS injection,compared with single LPS injection group(P=0.006＜0.05);however,treatments with rHDL52 and rHDL107 led to an increase of plasma IL-6(rHDL52:P=0.009＜0.01.rHDL107: P=0.02＜0.05,respectively).Similar to our observations at 24h after LPS injection,at 3h after LPS injection mice treated with rHDLwt,rHDL74 and rHDL52 had lower level of TNF-αthan those only received LPS injection(rHDLwt:P=0.049＜0.05,rHDL74:P=0.012＜0.05, rHDL52:P=0.048＜0.05,respectively).No significant alteration was observed in other groups of mice treated with rHDL107,rHDL129,rHDL173 and rHDL228 compared with LPS group.Recombinant HDL74,rHDL52 and rHDL107 could decrease the level of IL-1β,compared with LPS group(rHDL74:P=0.003＜0.05,rHDL52: P=0.007＜0.05,rHDL107:P=0.034＜0.05),while we did not see any significant effects of other rHDLs on reducing plasma IL-1β.None of the rHDLs showed a reduction of plasma IL-6 at 3h post LPS injection.Compared with 24h of LPS injection,the effect of rHDL on cytokine levels at 3 hours after LPS is biologically very small,suggesting a better neutralization of residual LPS in the circulation by rHDL injection for a longer time.Histological sections of lung tissueThe lung of mice only receiving LPS had significant pathological changes: 1)congestion.2) broadening of pulmonary interstitial tissue.3) leucocyte infiltration including monocytes and neutrophils,we observed that the mice treated with rHDLwt only exhibited slight pathological lung change,such as the moderate broadening of pulmonary interstitial tissue,compared with the saline group.In addition,the ability of rHDL74 to protect lung against LPS induced injury in this septic mice model is strongly supported by its histological results,which were very close to the saline group,and there was almost no pathological change in the Mut74 group.However,in lung sections from mice treated with rHDL228,we could not see any effect of reducing histological tissue injury,in stead of the aggravation of pathological changes, compared with LPS group.In addition,the histological sections of the other four rHDLs all had many pathological changes compared with the saline group.The weight ratios of DPPC and ApoA-I or ApoA-I mutants in rHDL assayThe weight ratios of DPPC to apoA-I in rHDL74 was slightly higher than in rHDLwt(1.93±0.06 vs.1.75±0.11).However,this ratios in rHDL228(0.82±0.05 vs.1.75±0.11,P＜0.05),rHDL107(1.49±0.12 vs.1.75±0.11,P＜0.05) and rHDL129 (1.47±0.04 vs.1.75±0.11,P＜0.05) were significantly lower than in rHDLwt.No significant alteration was observed in other rHDL groups.CONCLUSION:1.RHDL74 and rHDL52 are more effective than rHDLwt at reducing TNF-a and IL-1β.2.RHDL74 are more effective than rHDLwt at reducing IL-6.3.RHDL74 and rHDL52 are more effective than rHDLwt at attenuating endotoxin-induced clinical symptoms and protecting lung against LPS induced injury.4.Compared with rHDLwt,rHDL228 can significantly increase plasma concentration of TNF-αand exacerbate LPS induced lung injury.5.The weight ratios of DPPC to apoA-I in rHDL74 was slightly higher than in rHDLwt,However,this ratios in rHDL228 was the lowest among these rHDLs.In summary,for the first time,our study first compared the different effects of rHDL containing apoA-I cysteine mutants on the in vivo LPS induced endotoxemia in mice.Our data suggested that cysteine mutation not only induces the alteration of secondary structure and in vitro functions of apoA-I but also influences the capability of HDL to neutralize endotoxin or LPS.Due to its significant positive protection against LPS induced endotoxemia in our study,rHDL74 may be a potential clinical candidate for therapy of endotoxin induced septic shock.