Phsp70/IκBαm Prevents Acute Lung Injury Controllablly And Effectively

Background:Acute lung injury (ALI) and its severe form acute respiratory distresssyndrome (ARDS) are characterized by an overwhelming inflammatory processleading to non-specific disseminated damage of alveolar epithelium and vascularendothelium in the lung which can be caused by various of aetiological insults.The treatment of ALI/ARDS involves genearal supportive measures combinedwith lung protective ventilation and appropriate treatment of the underlyingconditions. Now there are no effective pharmacological therapies for ALI/ARDS.So ALI/ARDS remains a significant health burden with higher morbidity andmortality. Gene therapy is a promising candidate for the treatment of ALI/ARDS.However, which target gene is chosen and how to control the expression level oftarget gene according to need are the unsolved problem for gene therapy.Over-activation of NF-κB is the key point of the overwhelming inflammatoryprocess in ALI. NF-κB can specifically bind to the κB sequence in the promotersof many pro-inflammatory cytokines and promote their transcription andsynthesis. NF-κB also inhibits the apopotosis of neutrophils recruited to the lungand delay the fadeaway of the inflammation process. Therefore, NF-κB is one ofthe important target to regualte the inflammation response in ALI. Inphysiologcial condition, NF-κB binds to its inhibitor IκB and can not enter thenucleus, so its activity inhibited. IκB is phospholated (S32and S36) and degrated when the cell is stimulated by such as LPS, and the inhibition of IκBon NF-κB is released, so NF-κB enters the nucleus and promotes the productionof pro-inflammation cytokines. Mutant of IκBα (S32A and S36A) can not bephospholated and effectively inhibit the activity of NF-κB. However, NF-κB isan important transcription factor to maintain physiological function, totalinhibition of the activiy of NF-κB is also harmful. So one of the major aim inthis project is to establish a method to reactively adjust and control the activiy ofNF-κB according to the extent of inflammation response in lung inALI.Heat shock protein70(HSP70) is one of the important stress protein in cell.In ALI, overwhelming inflammation response causes a strong stress response inlung cells and induces high level of expression of HSP70. Previous studies haveshown that there is a positive correlation between the extent of inflammationresponse caused by lipopolysaccharide (LPS) and the expression level of HSP70protein. So the promoter activity of HSP70positively correlates with the extentof inflammation response caused by LPS. We hypothesize that the promoter ofHSP70can drive the expression of mutant IκBα(S32A/S36A) according to theextent of inflammation response caused by LPS, then reactively adjust andcontrol the activity of NF-κB and then the expression level of thepro-inflammatory cytokines.Aim and significance:Aim1) To construct a gene therapy vector named Phsp70/IκBαm in which HSP70promoter drives the expression of mutant IκBα (S32Aand S36A).2) To determine the provent effects of Phsp70/IκBαm on ALI in mice and thereactivity of Phsp70/IκBαm to the inflammation response caused by differentdoses of LPS. 3) To clarify whether Phsp70/IκBαm can reactively adjust and control theexpression level of IκBα protein then the activity of NF-κB and then thepro-inflammatory cytokines. We also compared the effect of HSP70promoter-drived mutant IκBα with that of CMV promoter-drived mutant IκBα.SignificanceThis project aims to construct a novel gene therapy vector namedPhsp70/IκBαm that can reactively adjust and control the activiy of NF-κBaccording to the extent of inflammation response in lung in ALI by driving theexpression of IκBα with HSP70promoter. The novel reactive vector will notover-inhibit the activity of NF-κB and maybe reduce the side effects of previousgene therapy vetors. It may afford new way to reactively adjust and control theexpression of target gene for gene therapy in ALI and other inflamatorydiseases.Method:Animal experimentsBALB/c mice (weight about18g, age4-6week) were randomly divided intofour groups (n=5): normal saline group, Phsp70/IκBαm group, LPS group andPhsp70/IκBαm plus LPS group. In normal saline group,24hours after micewere injected normal saline through tail vein, normal saline was administered byintratracheal instillation. In Phsp70/IκBαm group,24hours after Phsp70/IκBαmwas administered by tail vein injection, normal saline was administered byintratracheal instillation. In LPS group,24hours after mice were injected normalsaline through tail vein, different doses of LPS (0.3mg/kg,1mg/kg,3mg/kg) wasadministered by intratracheal instillation. In Phsp70/IκBαm plus LPS group,24hours after Phsp70/IκBαm was administered by tail vein injection, differentdoses of LPS (0.3mg/kg,1mg/kg,3mg/kg) was administered by intratracheal instillation.12h after intratracheal instillation, lung, heart and liver werecollected for detecting the expression level of IκBα and phosphorylated NF-κBprotein, pro-inflammation cytokines. The injury indices of wet to dry ratio oflung, MPO activity, protein and pro-inflammation concentration in BALF andmorphological change of lung were also measured.Cell experimentsA549or RAW264.7cells in exponential growth phase were cultured for18hin serum free medium, then Phsp70/IκBαm was transfected. The cells werestimulated with different of doses of LPS (0.01μg/mL,0.1μg/mL and1μg/mL)48h after being transfected. The mRNA level of IκBαand phopharylated proteinlevels of NF-κB were measured using RT-PCR and western blot. The LDHactivity, TNF-α and IL-6concentration in culture medium and the cell numberwere measured using ELISA and MTT respectively.Results:1. Phsp70/IκBαm can reactively prevent LPS-induced ALI according to theextent of inflammation response in mice.Phsp70/IκBαm significantly reduced the lung edema, the ratio of lung wet todry, ameliorated the pathological morphology change of lung tissue, reduced theactivity of MPO and the protein content in BALF. At the dose of0.01μg/mL,0.1μg/mL and1μg/mL LPS, the reduction of the ratio of lung wet to dry weightwas3.1%,16.9and39.1%, the inhibition of MPO activity and the proteincontent in BALF were4.3%,23.5%,52.3%and0.1%,5.7%,18.3%respectively.These results showed that the effect of Phsp70/IκBαm was positively correlatedwith the extent of inflammation response and the dose of LPS.2. Phsp70/IκBαm can reactively reduce LPS-induced inflammation responsein LPS dose dependent and negative feedback manner in mice. Transduction of Phsp70/IκBαm into mice significantly increased theexpression level of IκBα in lung. After LPS stimulation, the level ofphosphorylated NF-κB protein in lung and the concentration of TNF-α and IL-6in BALF significantly reduced in LPS dose dependent manner. At the dose of0.01μg/mL,0.1μg/mL and1μg/mL LPS, the reduction of the concentration ofTNF-α and IL-6in BALF was3.1%,16.9and39.1%respectively. The resultsshowed that Phsp70/IκBαm can reactively adjust the activity of NF-κB and theexpression of pro-inflammation cytokines.3. Phsp70/IκBαm can reactively ameliorate LPS-induced cell injury andinflammation response in a LPS dose dependent and negative feedback mannerin vitro.48h after Phsp70/IκBαm transfected into A549or RAW264.7cells, theexpression level of IκBα significantly increased, LPS stimulation furtherincreased the expression level of IκBα in a LPS dose dependent manner.Comparing with the negative control vector and CMV promoter-drived IκBαmutant, positive vector, Phsp70/IκBαm inhibited the activity of NF-κB in a LPSdose dependent manner. The negative control vector can not inhibit the activityof NF-κB whereas CMV promoter-drived positive vector significantly inhibitedthe activity of NF-κB but the inhibition effect was not correlated with the LPSdose.Phsp70/IκBαm significantly inhibited LPS-stimulated production of TNF-αand IL-6in A549and RAW264.7cells. The inhibition ratios was5.5%,17.8%,24.4%for TNF-α and0.5%,7.7%,20.8%for IL-6in A549cells, and10.1%,27.5%,46.4%for TNF-α and3.6.7%,27.6%,40.8%for IL-6in RAW264.7cellsat the dose of0.01μg/mL,0.1μg/mL and1μg/mL LPS. Nagative control vectorhad no significant inhibition on the production of TNF-α and IL-6but the inhibition effect of CMV promoter-drived positive vector was not correlatedwith LPS dose.Conclusions:1. Phsp70/IκBαm reactively adjusted and controled the expression of mutantIκBα (S32A and S36A), then inhibited the activity of NF-κB and then theproduction of the pro-inflammatory cytokines such as TNF-α and IL-6.2. Phsp70/IκBαm can reactively reduce LPS-induced inflammation response inLPS dose dependent and negative feedback manner in ALI mice.3. Phsp70/IκBαm can reactively ameliorate LPS-induced injury andinflammation response in a LPS dose dependent and negative feedback mannerin A549and RAW264.7in vitro.