Effects And Mechanisms Of Porphyromonas Gingivalis Lipopolysaccharide On Mitochondrial Function And Glycometabolism In Adipocytes

Objective3T3-L1 preadipocytes were cultured and differentiated in vitro.Porphyromonas gingivalis lipopolysaccharide(Pg-LPS)was used to simulate periodontal disease condition.To study the effects of Pg-LPS on mitochondrial morphology and oxidative phosphorylation function of adipocytes,the morphology of mitochondria was observed,the mitochondrial membrane potential,the content of cellular ROS and ATP,and the expression of mitochondrial dynamics related proteins were detected.Gene expression of mitochondrial biosynthesis related factors and mitochondrial DNA(mtDNA)copy number were detected to preliminarily explore the internal mechanism of mitochondrial damage in adipocytes.Glucose uptake of adipocytes was detected further in order to study the effect of Pg-LPS on glycometabolism of adipocytes.In this study,mitochondria were used as the entry point to reveal the connection between the toxic factor of periodontitis and mitochondrial damage of adipocytes,so as to provide a theoretical basis for in-depth research on the influence of periodontitis body glycometabolism.Metheds1.Cell culture:3T3-L1 preadipocytes were induced to adipogenic differentiation,and the differentiation status was identified by oil red O.2.Cell grouping:500 ng/mL and 1000 ng/mL Pg-LPS treatment group and blank control group were sampled at 12 h and 24 h after treatment.CCK8 was used to detect the cell viability.3.Mitochondrial morphology observation and oxidative phosphorylation function detection:Morphology of mitochondria was observed by transmission electron microscopy.Mitochondrial membrane potential and cellular ROS content were detected by flow cytometry.Cellular ATP content was detected by ELISA.Expression of mitochondrial dynamic related proteins Mfnl,Mfn2 and Drp1 was detected by Western Blot.4.Mitochondrial biosynthesis related factors and mtDNA assay:Gene expression of mitochondrial biosynthesis related factors PGC-1α,NRF1 and mtTFA,and mtDNA copy numbers were detected by fluorescence quantitative PCR.5.Glycometabolism detection:Glycometabolism of adipocytes was detected by flow cytometry.Results1.The 3T3-L1 preadipocytes successfully differentiated into mature adipocytes after 14 days of induction.2.After treatment of Pg-LPS,there was no difference in cell viability compared with the control group.3.Pg-LPS stimulation caused damage to mitochondrial morphology and function of adipocytes,as follows:①The appearance of mitochondria of adipocytes was swollen or wrinkled,the cristaes were broken and disordered.The basic structure of mitochondria disappeared after 24 h treatment of Pg-LPS.②The cellular ROS content was up-regulated in Pg-LPS treatment groups,but the results showed no statistical difference.③Compared with the control group,mitochondrial membrane potential and cellular ATP content in Pg-LPS treatment groups were significantly reduced.④Expressions of Mfn1,Mfn2 and Drpl were significantly up-regulated after 12 h of Pg-LPS stimulation.After 24 h of stimulation,Expressions of Mfn1 and Mfn2 were down-regulated.Drpl was significantly up-regulated in the 24 h-500 ng/mL group and significantly down-regulated in the 24 h-1000mg/mL group.4.Pg-LPS caused changes in mitochondrial biosynthesis related factor genes and mtDNA copy number,as follows:①In Pg-LPS treatment groups,the mRNA expression of PGC-1α was significantly up-regulated,while the mRNA expression of NRF1 and mtTFA was significantly down-regulated.②mtDNA copy number was significantly reduced in Pg-LPS treatment groups.5.Pg-LPS caused abnormal glycometabolism in adipocytes:In Pg-LPS treatment groups,the basal glucose uptake capacity of adipocytes was down-regulated,but the results showed no statistical difference.In the 24 h-1000 ng/mL group,the insulin-stimulated glucose uptake capacity of adipocytes was significantly down-regulated.Conclusion1.Pg-LPS can cause abnormal in mitochondrial morphology of adipocytes,disturb the normal fusion-fission balance of mitochondria,and damage its oxidative phosphorylation function.2.Pg-LPS can cause abnormal expression of mitochondrial biosynthesis related factors and mtDNA damage in adipocytes.3.High concentration of Pg-LPS stimulation can lead to abnormal glycometabolism in adipocytes.