Regulation Of PD-L1 By Autophagy In HPDLCs Under Hypoxic And Inflammatory Microenvironment

Objective:To explore the regulation of PD-L1 by autophagy in hPDLCs under hypoxic and inflammatory microenvironment.Method:(1)Culture the primary cells obtained from periodontal tissue.hPDLCs were cultured under normoxia,hypoxia,inflammation and the combination of hypoxia and inflammation for 24 h.The expression of PD-L1 in hPDLCs was detected by Western Blot,flow cytometry and confocal microscopy.(2)hPDLCs were cultured under the combination of hypoxia and inflammation for0 h,1h,3h,6h,12 h,24h.The expression of PD-L1 in hPDLCs was detected by Western Blot and confocal microscopy.(3)hPDLCs were cultured under the combination of hypoxia and inflammation for0 h,1h,3h,6h,12 h,24h.The expressions of p62/SQSTM1 and LC3 in hPDLCs were detected by Western Blot.(4)hPDLCs were cultured under the combination of hypoxia and inflammation for0 h,12h,24 h.The formation of autophagosomes and autolysosomes was observed by Transmission electron microscopy(TEM).(5)3-methyladenine and rapamycin were used as pharmacological agents to assess the regulation of PD-L1 by autophagy under hypoxic and inflammatory microenvironment.The expression of PD-L1 in hPDLCs was detected by Real-time PCR,Western Blot,flow cytometry and confocal microscopy.Results:(1)The expression of PD?L1 in hPDLCs cultured under the combination of hypoxia and inflammation was significantly lower than the hPDLCs cultured under inflammation(Western Blot: P<0.05;Flow cytometry: P<0.05;Mean luorescence intensity: P<0.01).(2)The expression of PD?L1 in hPDLCs cultured under the combination of hypoxia and inflammation was initially upregulated in a time-dependent manner from 0h to12h(Western Blot:P<0.001;Mean fluorescence intensity: P<0.001)but subsequently decreased from 12 h to 24h(Western Blot:P<0.05;Mean fluorescence intensity:P<0.001).(3)The ratio of LC3-II/LC3-I in hPDLCs cultured under the combination of hypoxia and inflammation was upregulated from 1h to 12h(Western Blot:P<0.01)and sustained in a high level from 12 h to 24 h.The expression of p62 was initially upregulated from 0h to 3h(Western Blot: P<0.05)but subsequently decreased from3 h to 24h(Western Blot:P<0.01).(4)Autophagosomes and autolysosomes were observed by TEM in hPDLCs cultured under hypoxia and inflammation for 12 h and 24 h.(5)The expression of PD?L1 in hPDLCs cultured under the combination of hypoxia and inflammation was significantly increased by 3-methyladenine(Real-time PCR:P<0.001;Western Blot:P<0.05;Mean fluorescence intensity:P<0.01).The expression of PD?L1 was significantly decreased by rapamycin(Real-time PCR:P<0.01;Western Blot:P<0.05;Mean fluorescence intensity:P<0.01);Flow cytometry analysis showed the expression of PD-L1 on the hPDLCs pretreated by 3-methyladenine tended to increase,although no statistical difference was found.The expression of PD-L1 on the hPDLCs pretreated by rapamycin was significantly decreased(P<0.01).Conclusion:The expression of PD-L1 in hPDLCs under hypoxic and inflammatory microenvironment was significantly lower than inflammatory microenvironment.The expression of PD-L1 in hPDLCs was negatively regulated by autophagy.