Research On The Mechanism Of Gingivitis Type "Shanghuo" Based On Th17/Treg Balance And Metabolomics

Objective According to Traditional Chinese Medicine(TCM),"Shanghuo",a common symptom in daily life,manifests a series of excessive heat syndrome based on the imbalance of yin and Yang in the human body.The swelling and pain of the gingival is a common symptom in"Shanghuo",which belongs to the category of"Yaxuan"in TCM.Based on the previous research of the project group,we speculate that"Shanghuo"may be related to the immune and metabolism disorder of the body.Taking Th17/Treg balance and metabolism as the breakthrough point,we explored the possible pathogenesis of gingivitis type"Shanghuo"from two aspects:animal experiment and clinical research.Methods(1)Mixing the heat syndrome model of dried ginger,monkshood root and cinnamon Decoction and the silk ligature gingivitis model to create gingivitis type“shanghuo”model.40 rats were divided into gingivitis type“Shanghuo”group(GFR+GIN),gingivitis model group(GIN),deficiency heat group(GFR)and normal control group(CN).After Intragastric administration of the decoction for Wistar rats 18 days,we use silk ligation in gingivitis model with high sugar for up to 30 days.On 30th day,the pathological conditions of gingival tissue with the right upper second molar of rats were observed.(2)The cytokines IL-2,TNF,IFN-γ,IL-4 and IL-10 were detected by CBA.RT-q PCR and Western-blot techniques were used to detect the expression of mRNA and protein of Foxp3,TGF-β,Rorγt in the spleen of the rats.The ELISA technique was used to detect the TGF-βand IL-17A secreted in the rat plasma.The percentage of CD4~+CD25~+Treg cells in whole blood and the percentage of CD3~+CD8~-IL-17A~+Th17 in PBMCs were detected by flow cytometry.(3)In clinical research,patients with gingivitis type of"Shanghuo"and healthy volunteers were recruiated.The mRNA expression of Foxp3,TGF-βand IL-17A in peripheral blood lymphocytes of two groups of people was detected by RT-q PCR technique.The ELISA technique was used to detect the TGF-βand IL-17A secreted in the plasma and the SIgA in saliva.Flow cytometry was used to detect the percentage of CD4~+CD25~+Foxp3~+Treg cells and the percentage of CD4~+IL-17A~+Th17 cells in whole blood in all groups.GC-MS metabolomics method was used to detect the plasma metabolites in the gingivitis type“Shanghuo”group.Statistical analysis was used to find out the plasma metabolites and related metabolic pathways in the gingivitis type“Shanghuo”group.Results(1)The GFR+GIN group rats have phenomenon of excessive heat,with messy hair,a slow increase of body weight,increased water intake,decreased urine volume,yellow color,less dry stool,not easy to grasp,even activly biting people and the local redness and swelling of the gums.The rat gingival pathology of GFR+GIN group showed that gingival epithelial hyperplasia,connective tissue vascular dilatation and congestion and a large amount of inflammatory cells.(2)Compared with the control group,the IL-2,IFN-γand TNF were slightly lower,while IL-4 and IL-10 were increased in the GFR+GIN group.The mRNA and the protein content of TGF-β,Rorγt,Foxp3 in the spleen of the rats in GFR+GIN group was the highest;the content of IL-17A and TGF-βin GFR+GIN group and GIN group was higher than in the CN group,and there were significant differences.Flow cytometry showed that the percentage of CD3~+CD8~-IL-17A~+Th17 cells in PBMCs of GFR+GIN group rats were the highest.The change of the percentage of CD4~+CD25~+Treg cells was not obvious.The percentage of Th17/Treg cell ratio,the GFR+GIN group was the highest with significant difference compared with the other three groups.(3)In clinical research,we recruited 27patients with gingivitis type"Shanghuo",which the excessive heat group(excessive stomach heat syndrome)20 cases,deficiency heat group(kidney yin deficiency syndrome)7 cases,and25 cases of healthy people(group HC)as control.Compared with the normal control group,the relative expression of TGF beta,IL-17A and Foxp3 in the patients with gingivitis type"Shanghuo"was significantly higher than that of the normal control group.The expression of IL-17A and TGF-βin the plasma of gingivitis type"Shanghuo"was significantly higher than that in the normal control group.The percentage of CD4~+IL-17A~+Th17 cells in patients with gingivitis was higher than that in the normal control group,and the difference was statistically significant,while the difference between the percentage of CD4~+CD25~+Foxp3~+Treg cells in the whole blood was not significant.As for the percentage ratio of Th17/Treg cells,the patients with gingivitis type"Shanghuo"were significantly higher than those in the normal control group.SIgA in the saliva of patients with gingivitis type"Shanghuo"was significantly higher than that in the normal group.There was a significant difference in the metabolic profiles of the serum from the gingivitis-type"Shanghuo"group and the normal control group,and 18 different metabolites were screened and identified including lactic acid,linoleic acid,arachidonic acid,glycine,glucose,cholesterol and so on.The main pathways affected were amino acid metabolism,glycolysis or gluconeogenesis,and fatty acid metabolism.Conclusion(1)In the animal research,the gingivitis type“Shanghuo”model has many similar signs with the gingivitis patients,so it can be used as animal model of gingivitis type“Shanghuo”.The cytokines related with Treg and Th17 and elevated Th17/Treg cell percentage ratio suggested the.imbalance of Th17/Treg in the gingivitis type“shanghuo”model.(2)In the clinical research,The cytokines related with Treg and Th17 and the increase in the percentage ratio of Th17/Treg cells suggests that the imbalance of Th17/Treg in the gingivitis type“shanghuo”.The level of SIgA in oral saliva of patients with gingivitis was increased,suggesting that local humoral immunity is enhanced.There were abnormalities in glycolysis,fatty acid metabolism,and amino acid metabolism in gingivitis type“shanghuo”group.(3)Based on the animal research and clinical research,we conclude that the imbalance of Th17/Treg and oral local immune disorders and abnormalities in glycolysis or gluconeogenesis,fatty acid metabolism,and amino acid metabolism pathways may be the mechanism in the pathogenesis of gingivitis type“Shanghuo”.