Lipopolysaccharide Prevents Methotrexate-induced Apoptosis In RAW264.7 Macrophage Cells

Sepsis is defined as a microbial infection(Gram-negative bacteria)that produces a series of systemic immune responses.Then,sepsis eventually leads to organ damage due to infections.Sepsis mortality had dropped to 15-25% because of the development of medicine.However,it is still an international healthy problem,needing global efforts to overcome togetherly.Thus,some of the old drugs are becoming novel points.Researchers focus on seeking possible drugs to therapy sepsis triggered by lipopolysaccharide(LPS).We have the honor to cooperate with the Johns Hopkins University of Medicine for screening drug inhibiting LPS canonical signaling pathways in their drugs libraries.Fortunately,we found that Methotrexate(MTX)could inhibit TLR-4,IRAK-4,I?B and NF-?B up-regulation expression induced by LPS on RAW264.7 cells.AT the same time,MTX displayed obvious cytotoxicity when it inhibits inflammatory responds induced by LPS.Therefore,this report focuses on how to avoid or decrease the side effects of MTX on therapy.MTX,an immune inhibitor,could inhibit dihydrofolate reductase to block the cell cycle and commonly used in tumor and autoimmune disease treatment.Yet,there are a variety of side effects,such as gastrointestinal reaction,stomatitis,bone marrow suppression,et al.To a certain extent,oral folic acid can prevent MTX-triggered cytotoxicity and other side effects when MTX is used for therapy in the clinical.We also found that low dose LPS can protect MTX-incduced cytotoxicity on the experiment.Then,we found that the protective function of LPS was related to apoptosis.And,we had further explored the molecular mechanisms of protective function of LPS on MTX,which were mediated via mitochondrial apoptosis protein P53 and Bcl-2 family other than canonical NF-?B.Therefore,the possible mechanisms exist in that low dose LPS prevent MTX-induced apoptosis,will be a novel method to reduce side effects for clinical rational use of MTX,such as folic acid rescue therapy.Similarly,it also provides a way to for prevent the side effects of MTX when it used to the treatment of sepsis in the future.Not only that,further study of low dose LPS prevention on MTX-induced cytotoxicity,can also give us a comprehensively perspective to know the basic physiological characteristics of LPS and provide a new research direction on LPS-related disease.Part?: LPS can inhibit MTX-induced cytotoxicityObjective: It is proved that optimal concentration a LPS protection of MTX-induced cytotoxicity.Methods:(1)MTX-induced toxic effects on RAW264.7 macrophages: firstly,0 uM,0.001 uM,0.001 uM,0.01 uM,1uM and 10 uM MTX stimulate RAW264.7 macrophages,secondly,use Alamar Blue to detect cell viability.(2)According to MTX toxicity,it was divided into following groups: blank control group,1u M MTX group,10 u M MTX group.And,LPS was diluted by 10 times into different concentration(0ng/ml,1ng/ml,10ng/ml,100ng/ml LPS)for intervention,respectively.After co-incubated for 24 hourss,use Alamar Blue to detect cell viability.(3)Identically,counting the number of cells under microscope,and extracting protein for testing total protein through BCA method after co-incubated for 24 hourss.(4)Collect data for analysis.Results:(1)Cytotoxicity began to appear when MTX reached 0.1uM(P < 0.01).(2)When MTX reached 1uM,10 uM,10ng/ml and 100ng/ml LPS could protect MTX-induced cytotoxic effect(P < 0.01).(3)When MTX reached 1uM,10 uM,10ng/ml and 100ng/ml LPS could prevent MTX-induced cell count and total protein decreasing(P < 0.01).Conclusion: 10ng/ml and 100ng/ml LPS can protect MTX-induced cytotoxicity.Part ?: LPS can inhibit MTX-induced apoptosisObjective: To prove cytotoxicity caused by apoptosis.Methods:(1)Experimental groups were divided according to the first part(2).And,each group was co-stimulated with LPS and MTX for 8 hours.Lysing the cells and detecting caspase-3 and cleavage caspase-3 expression levels via Western blotting.(2)According to MTX toxicity,experiment could be divided into three groups: blank control group,1uM MTX group,10 uM MTX group.And,each group was given different LPS concentration(0ng/ml,10ng/ml)for intervention.After co-incubated for 24 hourss,apoptosis was detected by Tunnel method and total number and percentage of Tunnel-positive cells were counted,respectively.(3)Collect data for analysis.Results:(1)MTX can up-regulate cleavage caspase-3 expression,but LPS can prevent MTX-induced cleavage caspase-3 up-regulation(P<0.01).(2)Tunnel staining showed that 10ng/ml LPS significantly reduced MTX-induced Tunnel-positive cells(P < 0.01).Conclusion: It that 10ng/ml LPS can prevent MTX-induced cytotoxicity is closely related to apoptosis.Part ?: The protection of LPS on MTX-induced apoptosis is little related to NF-?BObjective: Validate the correlation between the protection of LPS on MTX-induced apoptosis and NF-?B signal pathways.Methods:(1)Experimental groups were divided according to the first part(2).And,each group was co-stimulated with LPS and MTX for 8 hours.Cells were lysed and then Nitric Oxide Synthetase(NOS),NF-?B and phosphate-NF-?B expression levels were detected via Western blotting.Besides,IL-6 and Nitric Oxide(NO)in the supernatant were detected after co-incubation for 24 hours.Results: Compared with the blank control group,MTX cannot affect NF-?B and NOS.Meanwhile,inflammatory cytokines caused by LPS also has not been decreased(P > 0.05).Conclusion: NF-?B signal pathways may not involve in regulating the protection of LPS on MTX-induced apoptosis.Part ? : LPS prevents MTX-induced apoptosis via mitochondria related protein P53 and Bax/Bcl-2Objective: Validate the correlation between the protection of LPS on MTX-induced apoptosis and mitochondria related protein P53 and Bax/Bcl-2.Methods:(1)Experiment was divided as following: blank control group(C group),10ng/ml LPS group(L group),1uM MTX group(M group),10ng/ml LPS and 1uM MTX group(LM group).Co-stimulated with LPS and MTX for 24 hours,RAW264.7 cells were lysed and then P53,phosphate-P53 and Bax/Bcl-2 expression levels were detected through Western blotting.(2)Above all,experiment was divided into 4 groups: blank control group(C group),P53 inhibitors Pifithrin-? group(PF group),1uM MTX group(M group),Pifithrin-? and 1uM MTX group(PFM group).Co-incubated with LPS and MTX for 24 hours,objective cells were lysed and then P53,caspase-3 and cleavage caspase-3 expression levels were detected through Western blotting.(3)At last,experimental groups were divided according to the first part(2).And then,cells were co-stimulated with LPS and MTX for 24 hours.Mitochondrial changes in cytoplasm were observed through Transmission Electron Microscope(TEM).Results:(1)LPS prevents MTX-induced apoptosis via the down-regulation of P53 expression levels.P53 inhibitors Pifithrin-? can protect MTX-induced the up-regulation of cleavage caspase-3(P<0.01).(2)LPS prevents MTX-induced apoptosis through regulating Bax/Bcl-2 ratio(P<0.01).(3)TEM shows that LPS can prevent MTX-induced mitochondrial damage.Conclusion: LPS inhibits MTX-induced apoptosis via mitochondria related protein P53 and Bax/Bcl-2.