| Sepsis is an inflammatory response syndrome caused by infection.With the aggravation of the disease,multiple organ dysfunction and failure may even lead to death.In China,sepsis causes huge financial burden to the family,and has a high mortality rate.At present,the treatment strategy for sepsis is very limited and the therapeutic effect is not ideal.Therefore,it is important to find a new target for the treatment of sepsis.IFITM3(Interferon induced transmembrane protein 3)is a broad-spectrum antiviral protein.It participates in the regulation of a variety of physiological processes including innate immunity,cell adhesion,inflammation and so on.Recent studies have shown that Mycobacterium tuberculosis increases the expression of IFITM3 mRNA by acting on TLR4.IFITM3 can regulate the acid environment of lysozyme and inhibit the growth of Mycobacterium tuberculosis.It is suggested that IFITM3 not only participates in the regulation of the antiviral process,but also plays an important role in inhibiting the proliferation of bacteria.In addition,the abnormal expression of IFITM3 mRNA in schizophrenic patients suggests that IFITM3 may have a certain relationship with neurotransmitter acetylcholine.In periphery,The cholinergic anti-inflammatory pathway is an important way of immunomodulation for sepsis.Acetylcholine combined with alpha7 n acetylcholine receptor in macrophage will play a role of anti-inflammation and anti-apoptosis,but it is still unknown whether IFITM3 is involved in regulation of the cholinergic pathway.In this experiment,the relationship between IFITM3 and acetylcholine receptor was explored in RAW264.7,and the role of anti-inflammation and anti-apoptosis of IFITM3 in the cholinergic pathway has been verified.These findingswould provide some experimental basis for the next study on the mechanism of IFITM3 and the treatment strategy of sepsis.ObjectiveTo explore the relationship between IFITM3 and acetylcholine receptor in sepsis model and to verify the role of anti-inflammation and anti-apoptosis of IFITM3 in the cholinergic pathway.Materials and methods Part 1Experiment 1.RAW264.7 cell lines was divided into two groups: Control and LPS.LPS group was stimulated with 1 mg/ml LPS,and Control group was given with saline.Protein samples were collected at 24 h,48 h and 72 h,respectively.Protein level of IFITM3 in each group was detected by Western Blot.Experiment 2.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21 and LPS+GTS-21+ a-BGT.LPS+GTS-21 group was cholinergic agonist group.Given 1 mg/ml LPS stimulating for 24 h and then treated with 50 mM cholinergic receptor agonist GTS-21.LPS+GTS-21+a-BGT group was cholinergic antagonist group.100 nM cholinergic receptor antagonist a-BGT was given at 12 h after 1 m g/ml LPS stimulation and 50 mM cholinergic receptor agonist GTS-21 was added at 24 h.Protein samples were collected at 48 h.Protein level of IFITM3 in each group was detected by Western Blot.Part 2Experiment 1.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21 and LPS+GTS-21+ a-BGT.The intervention method was same as Experiment 2 in part 1.The levels of TNF-a and IL-1b were determined by Elisa.Experiment 2.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21 and LPS+GTS-21+a-BGT.The intervention method was same as above.The level of LDH was determined by colorimetric test.Experiment 3.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21 and LPS+GTS-21+a-BGT.The intervention method was same as above.The level of cell apoptosis was determined by TUNEL test.Part 3Experiment 1.RAW264.7 cell lines was divided into nine groups: Control(MOI=1),Control(MOI=10),Control(MOI=100),Virus(MOI=1),Virus(MOI=10),Virus(MOI=100),Virus+Polybrene(MOI=1),Virus+Polybrene(MOI=10),Virus+Polybrene(MOI=100).Control groups were given with saline.Virus groups were treated with virus that inhabited expression of IFITM3.Virus+Polybrene groups were treated with virus and Infective agent meanwhile.MOI=1,MOI=10,MOI=100 respectively represented the transfection ratio of vius and cell was 1:1,10:1,100:1.Transfection effect was observed by fluorescence microscope in 48 h.Experiment 2.RAW264.7 cell lines was divided into four groups: Negative,shRNA-1,shRNA-2,shRNA-3.Negative group was given with negative control shRNA-carrying lenti-virus.shRNA-1 group,shRNA-2 group and shRNA-3 group were three kinds of virus that constructed a vector with different IFITM3 shRNA.The protein level of IFITM3 was determined by Western Blot.Experiment 3.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21,LPS+GTS-21+Lv-shIFITM3.LPS+GTS-21 group was cholinergic agonist group.Given 1 mg/ml LPS stimulating for 24 h and then treated with 50 mM cholinergic receptor agonist GTS-21.LPS+GTS-21+Lv-shIFITM3 group was cholinergic agonist group but inhabited expression of IFITM3.50 mM cholinergic receptor agonist GTS-21 was given at 24 h and virus was given at 48 h after 1 mg/ml LPS stimulation.The levels of TNF-a and IL-1b were determined by Elisa at 72 h.Experiment 4.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21,LPS+GTS-21+Lv-shIFITM3.The intervention method was same as Experiment 3 in part 3.The level of LDH was determined by colorimetric test at 72 h.Experiment 5.RAW264.7 cell lines was divided into four groups: Control,LPS,LPS+GTS-21,LPS+GTS-21+Lv-shIFITM3.The intervention method was same as above.The level of cell apoptosis was determined by TUNEL at 72 h.ResultsPart 1Experiment 1.Compared with Control group,the protein level of IFITM3 was significantly decreased at 24 h,48 h and 72 h after 1 m g/ml LPS stimulation(P<0.01,P<0.01,P<0.01).Experiment 2.Compared with Control group,the protein level of IFITM3 in LPS group was markedly decreased(P<0.001).Compared with LPS group,the protein level of IFITM3 in LPS+GTS-21 group was markedly increased(P<0.001).while compared with LPS+GTS-21 group,the protein level of IFITM3 in LPS+GTS-21+?-BGT group was markedly decreased(P<0.001).Part 2Experiment 1.Compared with Control group,the levels of TNF-? and IL-1b in LPS group were significantly increased(P<0.01,P<0.01).Compared with LPS group,the levels of TNF-a and IL-1b in LPS+GTS-21 group were significantly decreased(P<0.01,P<0.01).While compared with LPS+GTS-21 group,the levels of TNF-a and IL-1 b in LPS+GTS-21+?-BGT group were notably increased(P<0.01,P<0.01).Experiment 2.Compared with Control group,the level of LDH in LPS group was obviously increased(P<0.05).Compared with LPS group,the level of LDH in LPS+GTS-21 group was obviously decreased(P<0.05).While compared with LPS+GTS-21 group,the level of LDH in LPS+GTS-21+?-BGT group was obviously increased(P<0.05).Experiment 3.Compared with Control group,the level of cell apoptosis in LPS group was obviously increased(P<0.01).Compared with LPS group,the level of cell apoptosis in LPS+GTS-21 group was obviously declined(P<0.01).While compared with LPS+GTS-21 group,the level of cell apoptosis in LPS+GTS-21+?-BGT group was obviously increased(P<0.01).Part 3Experiment 1.Compared with other groups,the transfection efficiency in Virus+Polybrene(MOI=100)group was best.Experiment 2.Compared with Negative group,the protein levels of IFITM3 in shRNA-1,shRNA-2 and shRNA-3 group were all declined(P<0.01,P<0.01,P<0.001).Experiment 3.Compared with Control group,the levels of TNF-a and IL-1b in LPS group were significantly increased(P<0.01,P<0.01).Compared with LPS group,the levels of TNF-a and IL-1b in LPS+GTS-21 group were significantly decreased(P<0.01,P<0.01).While compared with LPS+GTS-21 group,the levels of TNF-a and IL-1 b in LPS+GTS-21+ Lv-shIFITM3 group were not significant difference in statistics(P>0.05,P>0.05).Experiment 4.Compared with Control group,the level of LDH in LPS group was significantly increased(P<0.05).Compared with LPS group,the level of LDH in LPS+GTS-21 group was significantly decreased(P<0.05).While compared with LPS+GTS-21 group,the level of LDH in LPS+GTS-21+ Lv-shIFITM3 group was significantly increased(P<0.05).Experiment 5.Compared with Control group,the level of cell apoptosis in LPS group was obviously increased(P<0.01).Compared with LPS group,the level of cell apoptosis in LPS+GTS-21 group was obviously declined(P<0.01).While compared with LPS+GTS-21 group,the level of cell apoptosis in LPS+GTS-21+ Lv-shIFITM3 group was obviously increased(P<0.05).ConclusionOur results demonstrated that stimulating alpha7 n acetylcholine receptor can induce the increase in protein level of IFITM3 and play a role in anti-apoptosis via IFITM3.However,IFITM3 is not involved in regulating process of anti-inflammation. |
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