The Effect Of Total Platycodon Grandiflorus Polysaccharide Regulating M1 Type Polarization Of Porcine Alveolar Macrophages Mediates Via Autophagy

Platycodon grandiflorus(Jacq.)A.DC.(PG),the dry root of the Platycodon grandiflorus herbaceous plant,which is one of the most representative traditional Chinese medicines in China.PG has the effect of anti-inflammation and sedation,promote lung and expectorant,relieve pharynx,discharge abscess and reduce swelling.Its chemical components are polysaccharide,saponins,flavonoids,fatty oils,fatty acids,etc.Modern pharmacological studies have found that Platycodon grandiflorus has the effects of relieving cough and asthma,anti-oxidation,immune regulation,anti-inflammatory and antibacterial,anti-tumor,lowering blood lipid,lowering blood sugar,protecting liver,resisting lung injury and other effects.It is often used to treat symptoms such as cough and excessive sputum,poor chest tightness,dull sore throat,carbuncle and pus,and excessive phlegm.As one of the key bioactive components of the traditional Chinese medicine Platycodon grandiflorus,Platycodon grandiflorus polysaccharide has the effects of antioxidant,immune enhancement and anti-tumor.This study examined the surface morphology characteristics of Platycodon grandiflorus total polysaccharide(PGPS_t)and found that PGPS_t has a triple helical conformation,and has dense,porous and agglomerated morphological characteristics.By studying the effect of PGPS_ton autophagy of porcine alveolar macrophage cell line 3D4/21 cells,it was found that PGPS_tsignificantly up-regulated the expression of LC3-Ⅱand Beclin1,while the expression of P62was significantly reduced,suggesting that PGPS_t induced normal autophagic flux.immunofluorescence and transient transfection indicate that green fluorescent spots were generated around the nucleus after PGPS_t treatment,and they were clustered in dots.Transmission electron microscopy showed that autophagic vesicles with double membrane structure were found,indicating autophagosomes.After 3-MA pretreatment,the transformation of LC3-I type to type II and the degradation of P62 induced by PGPS_t were inhibited.The study of PGPS_t regulating the autophagy pathway of 3D4/21 cells found that PGPS_t mainly induces autophagy through PI3K/AKT/mTOR and MEK/ERK pathways,and the effect is strongest within 4-5 h.In 1-2 h,it mainly functions by activating the MEK/ERK pathway,and 3-5 h mainly by inhibiting the PI3K/AKT/mTOR pathway.These results indicated that PGPS_t can induce autophagy in 3D4/21 cells through PI3K/AKT/mTOR pathway and MEK/ERK pathway.Next,the regulation of PGPS_t on M1 polarization of 3D4/21 cells was studied.As a natural macromolecular active product,PGPS_t can regulate immunity and participate in the regulation of macrophage polarization.In order to study its regulatory effects,3D4/21 cells were treated with LPS/IFN-γto establish an M1 polarization model.The laser confocal microscope observed that the M1 cells were distributed in a circular shape.After testing,it was found that PGPS_tsignificantly promoted the mRNA expression of IL-6,IL-12 and TNF-αand promoted the expression of IL-6,IL-12,TNF-α,IL-1β,i NOS,CD80 and CD86 protein level.These results indicate that PGPS_t promotes the M1 polarization of 3D4/21 cells.SOCS1/2 is the regulatory protein in the JAK-STAT macrophage polarization pathway.In order to determine the effect of PGPS_t on M1 type polarization by regulating the expression of SOCS1/2 protein,it was found that PGPS_t significantly down-regulated the SOCS1/2 protein expression level,but has no obvious effect on the SOCS1/2 mRNA expression level.To explore the degradation pathway of SOCS1/2 protein,the proteasome inhibitor MG-132 and the lysosomal inhibitor CQ were used in conjunction with PGPS_t to detect the protein level of SOCS1/2.The results showed that after MG-132 treatment,PGPS_t can significantly reduce the expression level of SOCS1/2protein,while CQ significantly blocks the effect of PGPS_t on the expression of SOCS1/2 protein.The results showed that PGPSt degrades SOCS1/2 protein by activating the lysosomal system.Further research found that under the action of 3-MA,LC3BⅡinduced by PGPS_t was significantly reduced,and the down-regulation of SOCS1/2 protein by PGPS_t was blocked.Under the action of Baf A1,PGPS_t promotes the significant accumulation of LC3BⅡand P62,the autophagy flow is blocked,and the SOCS1/2 protein is no longer down-regulated by PGPS_t.The results showed that PGPS_t up-regulates LC3BⅡand down-regulates the expression of SOCS1/2 protein,which increased the possibility that LC3B,a key component of autophagy,bridged this connection and degraded SOCS1/2.To verify this possibility,the interaction between LC3 and SOCS2 was studied by indirect immunofluorescence and CO-IP.The results showed that after PGPS_t treatment,the colocalization percentage of the two proteins increased significantly,and there was an interaction between LC3 and SOCS1 and SOCS2 proteins.In conclusion,PGPS_t could induce 3D4/21 cell autophagy through PI3K/AKT/mTOR pathway and MEK/ERK pathway,and degraded SOCS1/2 protein through autophagy to promote M1 polarization of porcine alveolar macrophages.