| Purpose:Autoimmune thyroiditis is an organ-specific autoimmune disease mediated by T-lymphocytes,which is mainly manifested by a large number of T-cell infiltration in the thyroid gland and elevation of serum thyroid peroxidase antibody and thyroglobulin,and its clinical manifestations can be hyperthyroidism and hypothyroidism.Our research group has proved the efficacy of spleen tonification and qi supplementation in the treatment of AIT,and proposed that"spleen loss of guard"is the basis and premise of the pathogenesis of AIT,and innovatively summarized vitamin D as the subtle substances transported by the spleen.According to the clinical experience of our mentor,Prof.Gao Tianshu,Buzhong Yiqi decoction has good therapeutic efficacy for autoimmune thyroiditis patients with good safety and few side effects,but the mechanism of regulating the vitamin D metabolic pathway is not yet completely clear.In this paper,we took AIT model mice as research objects,and explored the pathogenesis of AIT and the mechanism of Buzhong Yiqi decoction in treating AIT through Vit D metabolism pathway from the aspects of histopathological changes of thyroid,serum antibody level,key enzymes of Vit D metabolism as well as the expression of VDR,RXR genes,DNA methylation,etc.,so as to provide the theoretical basis for the clinical use of AIT.Material and method:102 SPF-grade female NOD.H-2h4 mice,6-8 weeks old,23-27g,were acclimatized and fed for one week,and then divided into six groups by random number method:blank control group,model group,Buzhong Yiqi decoction high-dose group,Buzhong Yiqi decoction medium-dose group,Buzhong Yiqi decoction low-dose group,and western medicine group,with 17 mice in each group.Except for the blank control group,which was fed with distilled water,the other five groups were fed with 0.05%Na I solution for8 weeks.Pharmacological interventions were started from the 9th week of modeling,4.10g·kg-1Chinese medicine gavage per day in the BZL group,8.19g·kg-1Chinese medicine gavage per day in the BZM group,16.38g·kg-1Chinese medicine gavage per day in the BZH group,0.26mg·kg-1Western medicine gavage per day in the SS group,and 1 gavage per day in equal volume of distilled water in the NC group and the AIT group with 1 day rest for every 6 days of gavage,for 8 consecutive weeks.The resting period was 1 day for every 6days of gavage for 8 weeks.During the experiment,the mice were observed dynamically,and the serum,thyroid,spleen,liver and kidney tissues were taken from the mice at the end of the gavage.Experiment 1:HE staining and light microscopy were used to observe the histopathological changes in the thyroid gland of mice,and ELISA was used to detect the levels of Tg Ab,TSH,and 25（OH）D3 in mouse serum,flow cytometry was used to determine mouse spleen Th17,Treg cells;Experiment 2:q PCR was used to detect the expression of CYP2R1 and CYP27A1 in the liver,CYP27B1 m RNA in the kidney,and the expression of VDR,RXR,CYP27B1,CYP24A1 m RNA in the thyroid and the spleen;Western blotting was used to detect the expression of CYP2R1,CYP27A1 in the liver,renal CYP27B1 protein expression,thyroid and spleen VDR,RXR,CYP27B1,CYP24A1 protein expression;Experiment 3:q PCR assay for thyroid and spleen DNMT1,DNMT3a,DNMT3b m RNA expression,Western blot assay for thyroid and spleen DNMT1,DNMT3a,DNMT3b protein expression,and BSP assay for thyroid and spleen VDR,CYP27B1,CYP24A1 DNA methylation.Results:Experiment 1:1.Light microscopy showed a large number of follicle destruction,irregular morphology,and a large number of lymphocyte infiltration in the thyroid group of the AIT group mice,whereas the intervention of Buzhong Yiqi decoction and selenium improved the follicle destruction and the degree of lymphocyte infiltration to some extent compared with that of the AIT group.2.Results of the Tg Ab and TSH measurements:the levels of the serum thyroid antibody Tg Ab in the modeled mice were all significantly higher compared with that of the NC group,and the difference was statistically significant（P<0.001or P<0.05）,suggesting that the modeling of AIT mice had been successful.The difference was statistically significant（P<0.001 or P<0.05）,suggesting that the modeling of AIT mice was successful.Compared with the AIT group,the serum thyroid antibody Tg Ab levels of mice in the remaining treatment groups were reduced,and the difference was statistically significant（P<0.001）.The difference between the treatment groups was not statistically significant.Compared with the AIT group,the serum TSH levels of mice in the remaining groups showed a decreasing trend,but the difference between the groups was not statistically significant.3.25（OH）D3 Measurement Results:Compared with the NC group,the serum25（OH）D3 levels of mice in the remaining groups were reduced,with the AIT group being the most significant,and the difference was statistically significant（P<0.001）.Compared with the AIT group,the serum levels of 25（OH）D3 were increased in both the traditional Chinese medicine and western medicine treatment groups,and the differences were statistically significant（P<0.001 or P<0.05）.4.Flow cytometry results:compared with the NC group Th17/Treg of mice in AIT group increased most significantly.The levels of Th17/Treg in the traditional Chinese medicine and western medicine treatment groups were decreased,and the difference was statistically significant（P<0.001 or P<0.05）,especially in BZH group,the Th17/Treg reduction effect was most significant.Experiment 2:1.Spleen:Compared with the NC group,the spleen of mice in the AIT group had lower levels of Vit D metabolism-related genes CYP27B1,VDR and RXR m RNA（P<0.001）and higher levels of CYP24A1 m RNA（P<0.001）.The TCM group was able to elevate the spleen CYP27B1,VDR,and RXR m RNA levels and decrease the CYP24A1m RNA level in mice in the AIT group,with the best effect in the BZH group（P<0.001 or P<0.05）.The SS group was able to elevate the VDR m RNA level,but it was not statistically significant.Compared with the NC group,the protein levels of CYP27B1,VDR and RXR,key enzymes of Vit D metabolism in the spleen of mice in the AIT group were decreased（P<0.001）,and the protein level of CYP24A1 was increased（P<0.001）.The treatment group was able to increase the protein levels of CYP27B1,VDR and RXR and decrease the protein level of CYP24A1 in the spleen of mice in the AIT group（P<0.001）.2.Thyroid:Compared with the NC group,the levels of CYP27B1,VDR and RXR m RNA,key enzymes for Vit D metabolism in the thyroid gland of mice in the AIT group,were decreased（P<0.001）,and the level of CYP24A1 m RNA was increased（P<0.001）.The treatment group was able to elevate the thyroid CYP27B1,VDR,and RXR m RNA levels and decrease the CYP24A1 m RNA level in mice of the AIT group.Compared with the NC group,the protein levels of CYP27B1,VDR and RXR,key enzymes of thyroid Vit D metabolism,were decreased in the AIT group of mice（P<0.001）,and the protein level of CYP24A1 was increased（P<0.001）.The treatment group was able to elevate the protein levels of CYP27B1,VDR and RXR and decrease the protein level of CYP24A1 in the thyroid gland of mice in the AIT group（P<0.001）.3.Liver:Compared with the NC group,the m RNA levels of hepatic Vit D metabolism key enzymes CYP2R1 and CYP27A1 were decreased in the mice of AIT group（P<0.001）.m RNA levels were reduced（P<0.001）.Compared with the AIT group,all dose groups of traditional Chinese medicine and the SS group significantly increased the hepatic CYP2R1 m RNA levels of mice in the AIT group（P<0.001 or P<0.05）;the BZH group significantly increased the hepatic CYP27A1 m RNA levels of mice in the AIT group（P<0.001）,and there was a trend of increase in the CYP27A1 m RNA in the SS group but no statistical difference.Compared with the NC group,the protein levels of CYP2R1 and CYP27A1,key enzymes for Vit D metabolism,were reduced in the livers of mice in the AIT group（P<0.001）.Compared with the AIT group,both the TCM group and the SS group significantly elevated the hepatic CYP2R1 and CYP27A1 protein levels in mice in the AIT group（P<0.001 or P<0.05）,with no significant difference between the groups.4.Kidney:Compared with the NC group,the renal Vit D metabolism key enzyme CYP27B1 m RNA and protein levels in mice in the AIT group were reduced（P<0.001）The BZH,BZM and SS treatment groups were all able to significantly elevate the renal CYP27B1 m RNA and protein levels in mice of the AIT group（P<0.001 or P<0.05）.Experiment 3:1.DNA methyltransferase:Compared with the NC group,the m RNA levels of DNA methyltransferase DNMT3a and DNMT3b m RNA in the spleen of mice in the AIT group were elevated（P<0.001）.DNMT3a,DNMT3b m RNA levels were decreased in the treatment groups compared with the AIT group.Compared with the AIT group,DNMT1tended to decrease in all treatment groups,but there was no statistical difference.Compared with the NC group,the protein levels of DNA methyltransferase DNMT3a and DNMT3b were elevated in the spleens of mice in the AIT group（P<0.001）.Compared with the AIT group,DNMT1,DNMT3a and DNMT3b protein levels were decreased in each of the traditional Chinese medicine treatment groups and the SS group（P<0.001 or P<0.05）,and there was no significant difference between the groups.m RNA levels of thyroid DNA methyltransferase DNMT1,DNMT3a and DNMT3b m RNAs in the AIT group were elevated in the AIT group（P<0.001）,and the BZH and SS groups were able to significantly decrease the levels in the AIT group（P<0.001）,and the BZH group was able to significantly reduce the levels in the AIT group.m RNA levels of DNMT1,DNMT3a and DNMT3b were significantly reduced in the AIT group（P<0.001 or P<0.05）,with no significant difference between the two groups.Protein levels of DNMT1,DNMT3a and DNMT3b were elevated in the AIT group,and protein levels of DNMT1,DNMT3a and DNMT3b were elevated in the BZH group and SS group.were able to significantly reduce the protein levels of DNMT1,DNMT3a and DNMT3b in the thyroid gland of mice in the AIT group（P<0.001）,and there was no significant difference between the groups.2.VDR DNA methylation status:two Cp G islands were detected in the VDR gene of the spleen,and compared with the NC group,the VDR gene of the spleen was hypermethylated in the mice in the AIT group（P<0.001 or P<0.05）.The BZH group,BZM group,BZL group and SS group were able to reduce the DNA methylation level of VDR gene in AIT mice,and the difference between the BZH group and SS group in Cp G island 1 was statistically significant（P<0.05）.Thyroid VDR gene was detected in 2 Cp G islands,and the thyroid VDR gene was hypermethylated in mice in the AIT group compared with the NC group（P<0.001）,and the BZH group,the BZL group,and the SS group were able to reduce the level of thyroid VDR DNA methylation in AIT mice in Cp G island 1（P<0.001 or P<0.05）.The BZH group and the SS group in Cp G island 2 were able to reduce the methylation level of thyroid VDR DNA in AIT mice（P<0.05）.3.CYP24A1 DNA methylation status:2 Cp G islands were detected in the spleen CYP24A1 gene,and the spleen CYP24A1 gene was hypermethylated in the AIT group compared with the NC group in the Cp G island 1.Only the BZH group and the BZL group were able to reduce the CYP24A1 gene methylation level of AIT mice,but the difference between the groups was not statistically significant.The DNA methylation level of CYP24A1 gene was altered in each group in Cp G island 2,but the difference was not statistically significant.CYP24A1 gene DNA methylation levels were altered in Cp G islands 2 in the thyroid CYP24A1 gene,but the difference was not statistically significant.4.CYP27B1 DNA methylation status:2 Cp G islands were detected in the spleen CYP27B1 gene,and DNA methylation levels of CYP27B1 gene DNA methylation levels were altered among the groups,but the difference was not statistically Significance.Thyroid CYP27B1 gene detected 2 Cp G islands,compared with the NC group,the thyroid CYP27B1 gene was hypermethylated in mice of the AIT group（P<0.001）,and the SS group was able to reduce the DNA methylation level of the thyroid CYP27B1 gene of the AIT mice in Cp G island 1（P<0.001 or P<0.05）.The BZH group,the BZM group,and the SS group were able to reduce the DNA methylation level of the thyroid CYP27B1 gene of the AIT mice in Cp G island 2（P<0.001 or P<0.05）.group,BZH group and SS group in Cp G island 2 were able to reduce the DNA methylation level of thyroid CYP27B1 gene in AIT mice（P<0.001 or P<0.05）.Conclusion:1.Buzhong Yiqi decoction can improve the degree of thyroid lymphocyte infiltration and reduce the incidence of thyroid inflammation in AIT mice,Buzhong Yiqi decoction can reduce serum TGAb and TSH antibody levels,regulate immune balance in the Th17/Treg cell axis and elevate 25（OH）D3 levels in AIT mice;2.It suggests that Buzhong Yiqi decoction may effectively regulate the vitamin D metabolic pathway in AIT mice by up-regulating CYP27B1,VDR,RXR and down-regulating CYP24A1;Buzhong Yiqi decoction may affect the methylation level of VDR promoter region by regulating DNMTs,which in turn affects the vitamin D metabolic pathway in AIT mice. |
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