Role Of Perilipin 2 In Regulating Autophagy Of Macrophage Induced By Bacillus Calmette-Guérin Infection

Background:Tuberculosis(TB)is a zoonotic disease caused by Mycobacterium tuberculosis(Mtb)infection,which uses macrophages,an important immune cell in the body,as its main target cells.On the other hand,Mtb promotes macrophages to take in large amounts of fatty acids and store them in the form of lipid droplets,causing abnormal fatty acid metabolism in macrophages.It was found that Mtb can not only directly use lipids in lipid droplets to supply energy for its growth and reproduction,but also synthesize its own unique cell wall structure to guarantee the survival of Mtb.At the same time,the lipid molecules themselves and the key proteins in their metabolism affect autophagy and autophagy-related immune processes.Among them,lipid differentiation-associated protein 2(PLIN2),a member of the PAT family,is widely present in different species and is able to protect lipid droplets by blocking adipose triglyceride lipase(ATGL)and hormone-sensitive lipase(HSL),thus playing a key role in maintaining intracellular fatty acid metabolic homeostasis as well as lipid droplet formation and degradation.However,the role of PLIN2 in regulating cellular autophagy and fatty acid metabolism during Mtb infection of macrophages is not clear.Objectives and methods:In this experiment,mouse alveolar macrophages RAW264.7 were first used to knock down intracellular PLIN2 expression using small interfering RNA technology in combination with Mycobacterium tuberculosis vaccine strain Bacillus Calmette-Guerin(BCG)infection.Flow cytometry,Western blot and immunofluorescence observation were used to detect the differences in the expression of cellular autophagy rate,autophagy-related proteins and endoplasmic reticulum stress-related proteins,to clarify the role of PLIN2 in regulating fatty acid metabolism and cellular autophagy during BCG infection of macrophages from the in vitro level and to initially explore the mechanism.Subsequently,this experiment was conducted to construct a BCG-infected C57BL/6J mouse model by airway perfusion.At different stages after infection,the mice were executed and the differences in the expression of PLIN2 and autophagy-related proteins in mouse lung tissues were detected;then a model of interfering PLIN2-cooperative BCG infection was constructed by airway perfusion of PLIN2 small interfering RNA,and HE staining,immunohistochemistry and Western blot were used to detect the changes in lung tissue damage and autophagy-related protein expression,and to elucidate the effect of PLIN2 on BCG from the in vivo We used HE staining,immunohistochemistry and western blot to detect the changes of lung tissue damage and autophagy-related protein expression,and to elucidate the regulatory role of PLIN2 on autophagy in BCG-infected mouse lung tissue at the in vivo level.Results:1.BCG infection of macrophages significantly promoted PLIN2(p<0.05)and highly significantly upregulated the protein expression of autophagy-related factors ATG5,ATG12 and LC3(p<0.001),while inducing intracellular neutral lipid accumulation.Knockdown of PLIN2 significantly inhibited the protein expression of autophagy-related proteins ATG5 and ATG12(p<0.05)and LC3 and ATG7(p<0.001),and further revealed that it also significantly reduced the autophagy rate and intracellular neutral lipid content(p<0.001).2.After BCG infection of macrophages,knockdown of PLIN2 significantly inhibited the protein expression of endoplasmic reticulum stress-related protein CHOP(p<0.05)and highly significantly inhibited the protein expression of ATF4(p<0.001),and reduced intracellular Ca2+ concentration after BCG infection of macrophages.After continuous activation of the endoplasmic reticulum stress pathway using the endoplasmic reticulum stress agonist Tunicamycin,the expression of autophagy-related proteins ATG5 and ATG12 in BCG-infected macrophages was not significantly different with or without knockdown of PLIN2(p>0.05).3.BCG infection of C57BL/6J mice resulted in histopathological damage and highly significant promotion of lung tissue PLIN2 and autophagy-related proteins ATG5,ATG7 and ATG12 protein expression in lung tissue(p<0.001),which was positively correlated with the time of infection.Interfering with PLIN2 significantly increased the lung pathological damage in BCG-infected mice,and the expression of autophagy-associated proteins ATG7(p<0.01)and ATG12(p<0.001)in lung tissues was highly significantly reduced,and the lung bacterial load was significantly increased.Conclusion:BCG infection promoted the expression of PLIN2 in macrophages,which induced cellular autophagy by increasing intracellular fatty acid content,which in turn cascaded to activate the endoplasmic reticulum stress signaling pathway,thereby decreasing intracellular bacterial load and reducing BCG infection-induced lung injury in mice.