| Objective:Our team confirmed that "Bupi Yiqi" is the core treatment of Autoimmune thyroiditis(AIT)through clinical and experimental research.Based on the theory of"Piwei Zhiwei",this paper takes Dendritic cells(DCs)as the breakthrough point to study the therapeutic mechanism of Buzhong Yiqi decoctionin treating AIT.Through animal experiments,we observed the effect of Buzhong Yiqi decoctionon immune tolerance of spleen DCs in AIT mice.Through cell experiments,based on DNA methylation and PI3K-AKT-mTOR signaling pathway,the mechanism of Buzhong Yiqi decoction regulating DCs immune tolerance was revealed.Materials and methods:Paper one:The effect of Buzhong Yiqi decoctionon immune tolerance of spleen DCs in AIT mice102 SPF female NOD.H-2h4 mice were randomly divided into six groups after one week adaptive feeding:No-treatment Control(NG group),Model group(MG group),High-dose Buzhong Yiqi decoction group(BG-3 group),Middle-dose Buzhong Yiqi decoction group(BG-2 group),Low-dose Buzhong Yiqi decoction group(BG-1 group)and selenium group(SeG group).There were 17 mice in each group,of which 6 mice were used for HE,3 mice were used for flow cytometry and methylation,and 8 mice were used for ELISA.Except the NG group was fed with distilled water,the other five groups were fed with 0.05%NaI.At the eighth week of the experiment,serum and thyroid tissue were taken for detection.From the 9th week,the BG-1 group was given 4.10g/kg Buzhong Yiqi,the BG-2 group was given 8.19g/kg Buzhong Yiqi,the BG-3 group was given 16.38g/kg Buzhong Yiqi,the SeG group was given 0.26mg/kg sodium selenite,the NG group and MG group were given the same volume of distilled water.Every mouse takes the medicine once a day,rest for one day every seven days for eight weeks.We determine the incidence of thyroiditis and evaluate the degree of thyroid inflammation to observe the morphological of thyroid gland in mice by HE.The level of TgAb、TSH、25(OH)D and IL-10 in serum were detected by enzyme-linked immunosorbent assay(ELISA).To detect the expressions of CD40、CD80、CD86 and MHC-Ⅱ on the surface of DCs from spleen mononuclear cells by flow cytometry.Paper two:The DNA methylation mechanism of VDR gene regulated byBuzhong Yiqi decoction on DCs immune toleranceWe selected 40 SPF female SD rats in 11-week-old and randomly divided them into Chinese medicine group(23 rats)and No-treatment group(17 rats).The Chinese medicine group was given 8.19g/kg Buzhong Yiqi decoction(according to the optimal dosage in Paper one),while the No-treatment group was given the same volume of distilled water once a day for 7 days.Before the last administration,rats were fasted for 12 hours.One hour after the last administration,rats were anesthetized with 7%chloral hydrate.Blood was collected from abdominal aorta to prepare the medication and the myrrh of serum.The mouse bone marrow-derived dendritic cell strain was resuscitated and passaged until the cell density reached 80%.Cells were divided into No-treatment Control group(NG group),Buzhong Yiqi decoction group(BG group),Vitamin D group(VitD group)and Buzhong Yiqi decoction+Vitamin D group(BG+VitD group).Each group of the cells were collected for testing after 48 hours of intervention.The contents of VDR and IL-10 were detected by ELISA.Flow cytometry was used to detect the expression of CD40,CD80,CD86 and MHC-Ⅱ on the cell surface of each group.qPCR was used to detect the expression of VDR mRNA in DCs of each group.The expression of VDR mRNA and protein in DCs of each group were detected by qPCR and Westen Blot respectively.BSP was used to detect the DNA methylation of VDR gene in each group of DCs.Paper three:The regulatory mechanism of Buzhong Yiqi decoction onPI3K-AKT-mTOR signaling pathway of DCs immune toleranceThe dendritic cells after resuscitation were randomly divided into two parts.The first part is intervention by LY294002(the retardants of PI3K)which divided into No-treatment Control group(NG group),Buzhong Yiqi decoction group(BG group),Vitamin D group(VitD group),Buzhong Yiqi decoction+Vitamin D group(BG+VitD group),Buzhong Yiqi decoction+LY294002 group(BG+LY group)and Vitamin D+LY294002 group(BG+LY group).The second part is intervention by RAPA(the retardants of mTOR)which divided into No-treatment Control group(NG group),Buzhong Yiqi decoction group(BG group),Vitamin D group(VitD group),Buzhong Yiqi decoction+Vitamin D group(BG+VitD group),Buzhong Yiqi decoction+RAPA group(BG+RA group)and Vitamin D+RAPA group(BG+RA group).The cells in each group were collected after 24 hours and 48 hours of intervention respectively.The morphological changes of DCs in each group were observed dynamically by optical inverted phase contrast microscope.Flow cytometry was used to detect the expression of CD40,CD80,CD86 and MHC-II on the cell surface of each group.qPCR was used to detect the levels of PI3K,AKT and mTOR mRNA in each group.The protein levels of PI3K,p-PI3K,AKT,p-AKT,mTOR and p-mTOR were detected by Westen Blot.Results:Paper one:The effect of Buzhong Yiqi decoction on immune tolerance of spleen DCs in AIT mice1 Pathological changes of thyroid tissueIn NG group,the thyroid gland structure was complete and regular under light microscope,and the follicles were round or oval,and the size was relatively consistent.The follicles were filled with reddish colloid,and there was no lymphocyte infiltration.In MG group,the thyroid tissue structure was destroyed under light microscope,and the size of follicles was different.Most of the follicles were filled with colloid,some of them were missing colloid,follicles collapsed and shrank,and a large number of lymphocytes infiltrated in follicular cavity and interstitial tissue.Follicles of different sizes can be seen in the thyroid gland of other groups under light microscope,some follicles are filled with colloid,a few cavities are missing colloid,and lymphocytes can be infiltrated in the stroma to varying degrees,which is lighter than that of MG group,among which the middle dose group of traditional Chinese medicine has the most remarkable curative effect.2 Evaluation of AIT incidence and inflammation degree in mice of each groupThere was no lymphocyte infiltration in thyroid gland and the score of inflammation degree was 0+,and no autoimmune thyroiditis occurred in 6 mice in NG group,BG-2 group and SeG group.The infiltration area of thyroid lymph in 6 mice was about 30～50%,and the inflammation score was 3+,all of them developed AIT in MG group.There were 3 mice showed lymphocyte infiltration in thyroid gland,the infiltration area was about 1～10%,and the inflammation score was 1+in BG-3 group.Lymphocyte infiltration in thyroid gland was observed in 4 of 6 mice in BG-1 group,the infiltration area was about 1～10%,and the inflammation score was 1+.3 The changes of thyroid function in each groupCompared with NG group,the levels of serum thyroid antibody TgAb in model mice were significantly increased,and the difference was statistically significant(p<0.001 orp<0.05),suggesting that AIT mice were successfully established.Compared with MG group,the levels of serum thyroid antibody TgAb in other treatment groups were decreased,and the difference was statistically significant(p<0.001)especially in BG-2 group.There was no significant difference between BG-2 and BG-3(p=0.070).Compared with NG group,the TSH level of other groups increased,but the difference was not statistically significant.4 Vitamin D level of mice in each groupCompared with NG group,the vitamin D level in serum of other groups were decreased with statistical significance(p<0.001),especially in MG group.Compared with MG group,the vitamin D level of mice in other treatment groups were increased(p<0.001 or p<0.05).However,there was no significant difference between the treatment groups of traditional Chinese medicine(compared with BG-3 group and BG-2 group,p=0.622;compared with BG-3 group and BG-1 group,p=0.912;compared with BG-2 group and BG-2 group,p=0.125).5 The changes of DCs in spleen of each group5.1 The secretion of IL-10 in DCsCompared with NG group,the secretion of IL-10 in the spleen of MG group was decreased obviously(p<0.001).Compared with MG group,the level of IL-10 in spleen of other treatment groups was increased(p<0.001),but there was no statistical significance between BG-2 and BG-3 groups(p=0.831).5.2 The expression of CD40,CD80,CD86 and MHC-Ⅱ in DCsCompared with NG group,the surface factors of DCs in the spleen were increased(p<0.001 or p<0.05).Compared with MG group,the level of surface factors in DCs were decreased with statistical significance(p<0.001),but there was no statistical significance between BG-2 and BG-3 groups(CD40:p=0.739;CD80:p=0.496;CD86:p=0.511;MHC-Ⅱ:p=0.895).Paper two:The DNA methylation mechanism of VDR gene regulated by Buzhong Yiqi decoction on DCs immune tolerance1 The changes of DCs function1.1 The secretion of IL-10 in DCsCompared with NG group,the secretion of IL-10 in DCs in other groups were increased with statistical significance(p<0.001).The level of IL-10 in VitD group and BG+VitD group were lower than that in BG group(p<0.001).Although the level of IL-10 in BG+VitD group was higher than that in VitD group,there was no significant difference between them(p=0.100).1.2 The expression of CD40,CD80,CD86 and MHC-Ⅱ in DCsCompared with NG group,the expressions of CD40,CD80,CD86 and MHC-Ⅱon DCs in each group were decreased(p<0.001),especially in BG group.Compared VitD BG group,the level of DCs surface factors in other groups were increased slightly(p<0.001 or p<0.05,CD40:p=0.062).Although the levels of surface factors in VitD group were lower than those in BG+VitD group,there was no significant difference between the two groups(CD40:p=0.073;CD80:p=0.504;CD86:p=0.342;MHC-Ⅱ:p=0.521).2 The expression of VDR on DCs surface2.1 The content of VDR on DCs surfaceCompared with NG group,the level of VDR on DCs in other groups were increased(p<0.001),but there was no statistical difference among these groups(VitD group v.s.BG group:p=0.507;BG+VitD group v.s.BG group:p=0.263;VitD group v.s.BG+VitD group:p=0.083).2.2 The expression of VDR mRNA on DCs surfaceCompared with NG group,the level of VDR mRNA in each group were increased(p<0.001).Among these groups,the increase in BG+VitD group was the most obvious,and the difference between BG group and VitD group was statistically significant(p<0.001 orp<0.05).Although the level of VDR mRNA on DCs in VitD group was slightly higher than that in BG group,there was no significant difference between them(p=0.987).2.3 The expression of VDR protein on DCs surfaceCompared with NG group,the level of VDR protein in other groups were increased with statistical significance(p<0.001 or p<0.05).The level of VDR protein in BG+VitD group was higher than that in BG group and VitD group(p<0.05).There was no significant difference between BG group and VitD group(p=0.193).2.4 The methylation of VDR gene on DCs surfaceAfter the intervention of Chinese medicine and VitD on DCs,the promoter region DNA of VDR gene was hypomethylated,and the methylation degree of each group was significantly lower than that of NG group(p<0.001).The methylation of BG+VitD group was higher than that of BG group and VitD group,and the difference was statistically significant(p<0.001 orp<0.05).Although the DNA methylation of VDR gene was reduced in VitD group most obviously,there was no significant difference between BG group and VitD group(p=0.120).Paper three:The regulatory mechanism of Buzhong Yiqi decoction on PI3K-AKT-mTOR signaling pathway of DCs immune tolerance1 The effection of Buzhong Yiqi decoction on DCs1.2 The effect of Buzhong Yiqi decoction on DCs MorphologyCultured in an incubator with 5%CO2 at 37℃ for 0h,24h and 48h respectively,and the morphological changes of DCs in each group were observed.Under the inverted phase contrast microscope,there are fewer adherent cells and more suspended cells in all groups,and there are cell colonies at 0h.The cells are round or oval,with tiny pseudopodia at the edge and no dendrite formation.Cultured after 24h,it can be seen that the cells are scattered,most of them grow in suspension.The cells are star-shaped or spindle-shaped,the surface protrusions are different in thickness and size,and there are many branches like branches,which indicates that the cell activity is high at this time.Cultured after 24h,the number of cells were increased and almost all cells were surrounded by dendritic processes with irregular shapes.However,some cells died in the six groups of cells intervened by retardation,and the number of cells were less than that in other groups,indicating that the retardation had a certain killing effect on cells.1.3 The effection of Buzhong Yiqi decoction on DCs immune tolerance.Compared with NG group,the levels of CD40,CD80,CD86 and MHC-Ⅱ on DCs in BG group,VitD group and BG+VitD group were decreased,and the differences among other groups were statistically significant(p<0.001 orp<0.05)except for CD40 in BG+VitD group at 24h(p=0.219).Compared with BG+VitD group,the levels of CD40,CD80,CD86 and MHC-Ⅱ in BG group and VtiD group were decreased,and there were significant differences in all indexes of BG group and the level of CD40 in VitD group for 24 hours(p<0.001 orp<0.05.BG group v.s.BG+VitD group,after 24 hours of intervention,CD80:p=0.360,CD86:p=0.801,MHC-Ⅱ:p=0.692;after 48 hours of intervention,CD40:p=0.073,CD80:p=0.504,CD86:p=0.342,MHC-Ⅱ:p=0.521).Combined with LY294002 intervention for 24 hours and 48 hours respectively,the levels of CD40,CD80,CD86 and MHC-Ⅱ in BG group,VitD group and BG+VitD group were all decreased,and the difference was statistically significant(p<0.001).The result is similar to that of LY294002 group,the levels of CD40,CD80,CD86 and MHC-Ⅱ in BG group,VitD group and BG+VitD group were decreased after 24 hours and 48 hours of intervention by RAPR respectively(p<0.001 or p<0.05).2 The levels of PI3K,AKT and mTOR mRNA in DCs of each groupCompared with NG group,the levels of PI3K,AKT and mTOR mRNA in BG group,VitD group and BG+VitD group were increased(p<0.001).Compared with BG+VitD group,the levels of PI3K,AKT and mTOR mRNA in BG group and VtiD group were decreased,but there was no significant difference in AKT mRNA level in VitD group(p<0.001 orp<0.05.24h:p=0.153;48h:p=0.122).After 24 hours and 48 hours of combined intervention with LY294002,the levels of PI3K,AKT and mTOR mRNA in DCs of BG group,VitD group and BG+VitD group were increased(p<0.001 orp<0.05).Similar to LY294002 group,the levels of PI3K,AKT and mTOR mRNA in BG group,VitD group and BG+VitD group were increased after 24h and 48h of intervention by RAPA respectively(p<0.001 or p<0.05).3 The proteins levels of PI3K,AKT and mTOR in DCs of each groupAfter treatment with LY294002,compared with NG group,the phosphorylation levels of BG group,VitD group and BG+VitD group were increased(p<0.001).Compared with BG+VitD group,the protein levels of p-PI3K,p-AKT and p-mTOR in BG group and VitD group were decreased after the culture for 24 hours.After the culture for 48 hours,the protein level of p-PI3K was decreased and the protein level of p-AKT was increased in BG group and VitD group,but the protein level of p-mTOR was higher in BG group than in BG+VitD group and lower in VitD group(p<0.001 orp<0.05).Compared with the combined intervention of LY294002,the protein levels of p-PI3K,p-AKT and p-mTOR in BG group,VitD group and BG+VitD group were respectively higher than these in BG+LY group,VitD+LY group and BG+VitD+LY group.Meanwhile,the differences were statistically significant(p<0.001 or p<0.05)except for the p-AKT(p=0.125)、p-mTOR(p=0.111)after 24 hours and the p-PI3K(p=0.085)after 48 hours of intervention.After 48 hours of intervention by RAPA,the protein level of PI3K in VitD group and BG+VitD group was higher than that in NG group,while the protein level in BG+VitD group was lower than that in BG+VitD+RA group(p<0.001 or p<0.05).The protein level of mTOR in VitD group was significantly higher than that in VitD+RA group(p<0.05),but there was no statistical difference among other proteins.Compared with NG group,the phosphorylation levels of BG group,VitD group and BG+VitD group were increased and the differences among other proteins were statistically significant(p<0.001 orp<0.05)except for p-PI3K(p=0.300)in VitD group after 24 hours,p-PI3K(p=0.054)and p-AKT(p=0.296)in BG+VitD group after 48 hours.Compared with BG+VitD group,the protein level of p-PI3K,p-AKT and p-mTOR in BG group and VitD group were changed after 24 hours of culture.Except for the protein level of p-mTOR(p=0.345)in VitD group after 24 hours and the protein level of p-AKT(p=0.149)in BG group after 48 hours of intervention,there were differences among other protein groups(p<0.001 orp<0.05).Compared with RAP A,the proteins level of p-PI3K,p-AKT and p-mTOR proteins on DCs in BG group,VitD group and BG+VitD group were respectively higher than these in BG+RA group,VitD+RA group and BG+VitD+RA group,except for p-mTOR(p=0.245)in BG+VitD group after 48 hours,the differences in other indexes were statistically significant(p<0.001 orp<0.05).Conclusion:1.Buzhong Yiqi decoction can reduce the incidence of thyroid inflammation in AIT mice,improve the infiltration degree of thyroid lymphocytes in AIT mice,reduce the level of serum TgAb antibody,increase the level of 25(OH)D.Meanwhile,Buzhong Yiqi decoction can promote the secretion of IL-10 in spleen,reduce the expression of CD40,CD80,CD86 and MHC-II molecules on the surface of DCs derived from spleen monocytes in mice.The important thing is Buzhong Yiqi decoction can make DCs in immune tolerance state.2.Buzhong Yiqi decoction can inhibit the DNA methylation of VDR gene on DCs.It also can promote the secretion of IL-10 by DCs,reduce the expression of CD40,CD80,CD86 and MHC-II on DCs surface,and maintain DCs in immune tolerance.3.Buzhong Yiqi decoction can not only increase the levels of p-PI3K,p-AKT and p-mTOR mRNA and protein in DCs treated with LY294002 and RAPA,but also decrease the expressions of CD40,CD80,CD86 and MHC-II in DCs treated with retarders,suggesting that Buzhong Yiqi decoction can activate PI3K-AKT-mTOR signaling pathway in DCs and maintain DCs in immune tolerance. |
PDF Full Text Request