| Objective To evaluate the clinical curative effect of Tuina combined with acupuncture in the treatment of Hashimoto’s thyroiditis(HT)based on the theory of "Guben Tongjing".To explore the possible mechanism of this method in the treatment of HT in terms of immune regulation.To provide scientific basis and feasible plan for the treatment of HT with Tuina combined with acupuncture,and enrich the content of clinical application of external treatment of traditional Chinese medicine.MethodsThe combination of clinical controlled research and animal experiment verification is adopted.Observing the clinical curative effect of Tuina combined with acupuncture in the treatment of HT under the guidance of the "Guben Tongjing" theory through clinical controlled research,and discussing the changes in the physiological curvature of the cervical spine and the influence of factors such as temperature and emotion on the pathogenesis and treatment of HT;and then verifying the effectiveness of this method through animal experiment by further observing the effect of this method on inflammatory injury and TLR4/My D88/NF-κB signaling pathway in HT rat model.The specific method is as follows:1.Clinical researchA randomized control design was used to collect 68 HT patients who met the inclusion criteria from the Thyroid Department of Huanggang Hospital of Traditional Chinese Medicine.They were random Ly divided into a Western medicine treatment group(Selenium Yeast Tablet group)and a Tuina combined with acupuncture treatment group(Guben Tongjing group),34 cases in each group.For Selenium Yeast Tablets Group: each patient took one Selenium Yeast Tablet in the morning on an empty stomach once per day,for 8consecutive weeks of treatment.For the Guben Tongjing group: Under the guidance of "Guben Tongjing" theory,the patients were treated with Tuina combined with acupuncture,1 times every two days,10 times a course of treatment,2 days rest after each course of treatment,for 3 consecutive courses.Statistics,including the TCM syndrome scores,TPOAB,TGAB,IL-6,IL-12,goiter and blood flow,Cervical spine physiological curvature(X-ray),ΔT and SAS/SDS of the patients in each group,were separately recorded both before and after the treatments.And the results were statistically analyzed after the treatment.2.Experimental StudyForty 6-week-old SPF female SD rats were random Ly divided into blank control group(10 rats)and model group(30 rats).The 30 experimental rats in the model group were subcutaneously immunized with b Tg and FA,and fed with high iodine water(0.64g/L)to induce the HT rat model.After the 8th week of modeling,30 rats in the model group were random Ly divided into model control group,western medicine treatment group(Selenium Yeast Tablet group),Tuina combined with acupuncture treatment group(Guben Tongjing group),10 rats in each group.Together with the blank control group(10 rats)without modeling,all experimental rats were divided into 4 groups.Intervention started from the 9th week.The blank group was fed with purified water and normal saline at a dose of 1m L/100g;the model group was fed with high iodine water(0.64g/L)and a dose of 1m L/100 g normal saline;the Selenium Yeast Tablet group was fed with Selenium Yeast Tablet solution with concentration of 0.1 mg/L by 1m L/100 g once per day,for 6 consecutive weeks;the Guben Tongjing group was treated with Tuina combined with acupuncture(the treatment method was the same as was used in clinical research),and normal saline was administered at a dose of 1m L/100 g,5 times a week,10 times a course of treatment,2 days rest after each course of treatment,for 3consecutive courses(6 weeks).Specimens were collected after the end of the experiment at week 15.The concentration of TGAB and TPOAB,and the content of IL-6 and IL-12 of the rats in each group were tested through ELISA;the pathological changes of thyroid tissue in rats in each group were observed and scored through H&E;RT-PCR was applied to detect the m RNA expressions of TLR4,My D88,and NF-κB p65 in the thyroid tissues of rats in each group;WB and IHC were applied to test the protein expressions of TLR4,My D88,and NF-κB p65 in the thyroid tissues of rats in each group,and to statistically analyze the results.Results1.Clinical research(1)Clinical data and results: the number of included patients shows that there was no statistical difference between the two groups in terms of age,gender,and duration of onset(P>0.05).The he case dropouts of the enrolled patients were as follows: 1 case dropped out in the Selenium Yeast Tablets group,2cases dropped out in the Guben Tongjing group,and the case completion was relatively good;the total compliance was 89.32%,which was greater than the80.00% compliance criteria set by the study.It can be considered that good compliance has been obtained.(2)Comparison of clinical efficacy: In terms of TCM syndrome scores,the clinical symptoms of the two groups of patients were improved after treatment and during the follow-up period,and the comparison within the group was statistically significant(P<0.05),and the Guben Tongjing group had a much more significant improvement in symptoms than the Selenium Yeast Tablet group(P<0.05).The total effective rate of the Guben Tongjing group was90.6%,which is better than that of the Selenium Yeast Tablet group,the effective rate of which was 66.67%(P<0.05).(3)Comparison of concentrations of TGAB and TPOAB: The concentrations of TGAB and TPOAB decreased in the two groups after treatment and during the follow-up period,and the difference was statistically significant(P<0.05).After treatment and during the follow-up period,the decrease of the concentration levels of TGAB and TPOAB in the Guben Tongjing group was greater than that in the Selenium Yeast Tablet group,and the difference was statistically significant(P<0.05).(4)Comparison of the IL-6 and IL-12 contents: the serum IL-6 and IL-12 contents of the two groups of patients decreased after treatment and during the follow-up period,and the difference was statistically significant in the comparison within the group(P<0.05).After treatment and during the follow-up period,the Guben Tongjing group showed a greater decrease in the serum IL-6 and IL-12 contents than the Selenium Yeast Tablet group,and the difference was statistically significant(P<0.05).(5)Comparison of thyroid volume and isthmus thickness: The thyroid volume(V)and isthmus thickness of the two groups generally decreased after treatment and during the follow-up period,and the difference was statistically significant in comparison within the group(P<0.05).The thyroid volume(V)and isthmus thickness showed a larger decrease in the Guben Tongjing group after treatment and during the follow-up period than in the Selenium Yeast Tablet group,and the difference was statistically significant(P<0.05).(6)Correlation between thyroid hemodynamics and cervical curvature:Pearson linear correlation analysis showed that there was a weak positive correlation between the inner diameter of the superior thyroid artery and cervical curvature(r=0.1486,P=0.1360),and there was no statistical correlation Scientific significance(P>0.05).The peak flow velocity,average flow velocity and resistance index of superior thyroid artery were weakly positively correlated with the changes of cervical physiological curvature(r=0.2738,0.3220,0.2716),and the correlation was statistically significant(P<0.05).(7)Correlation between thyroid antibody levels and cervical curvature,temperature,and emotion: Pearson linear correlation analysis showed that the concentration levels of TGAB and TPOAB were positively correlated with cervical curvature(r=0.2421,0.2981),and the correlation was statistically significant(P<0.05);the concentration levels of TGAB and TPOAB were positively correlated with local temperature changes of the thyroid gland(r=0.4835,0.522),and the correlation was statistically significant(P<0.05);the concentration levels of TGAB and TPOAB were positively correlated with emotional changes(r=0.621,0.7397,0.4802,0.7934),the correlation was statistically significant(p<0.05).2.Experimental Study(1)General conditions of the rats: After the modeling was completed,none of the rats died.The TPOAB levels of the rats in the model group were higher than 60IU/m L as was measured through ELISA,and inflammatory infiltration was seen in the thyroid pathological examination,which shows that the modeling was successful and that it met experimental requirements.During the experimental intervention,one rat died in each of the two groups,and one rat in the selenium yeast tablet group was eliminated.The number of rats in each group was: 10 rats in the blank group,10 rats in the model group,8 in the selenium yeast tablet group,and 9 in the Guben Tongjing group.And the number of experimental rats in each group had no statistical difference(p>0.05),which was comparable.(2)TGAB and TPOAB results: compared with the blank control group,TGAB and TPOAB concentration of rats in the model control group increased,and the difference was statistically significant(p<0.05);compared with the model control group,TGAB and TPOAB concentration generally declined in both the Selenium Yeast Tablet group and the Guben Tongjing group,and the difference was statistically significant(p<0.05).Moreover,it showed a larger decrease in the Guben Tongjing group than in the Selenium Yeast Tablet group,and the difference was statistically significant(p<0.05).(3)IL-6 and IL-12 Results: compared with the blank control group,the IL-6and IL-12 level of the rats in the model control group increased,and the difference was statistically significant(p<0.05);Compared with the model control group,the IL-6 and IL-12 level decreased in both the Selenium Yeast Tablet group and the Guben Tongjing group,,and the difference was statistically significant(p<0.05);compared with the Selenium Yeast Tablet group,the IL-6 and IL-12 level of the rats in the Guben Tongjing group were lower than that in the Selenium Yeast Tablet group,and the difference was statistically significant(p<0.05).(4)H&E results: the pathological structure of the thyroid gland in the blank control group was basically normal,and there was no obvious inflammatory cell infiltration in the tissue;the pathological structure of the thyroid gland in the model control group was slightly abnormal,and some inflammatory cells was detected in the follicular colloid;the pathological structure of the thyroid gland in the Selenium Yeast Tablet group was slightly abnormal,and some inflammatory cells appeared in the follicular colloid;the pathological structure of the thyroid gland in the Guben Tongjing group was slightly abnormal,and there was some inflammatory cells in the follicular colloid.Inflammation scores showed that the reduction rate of thyroid tissue inflammation scores in the Guben Tongjing group was larger than that in the model control group and that in the Selenium Yeast Tablet group,and the difference was statistically significant(P<0.05).(5)RT-PCR results: compared with the blank control group,the m RNA expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of rats in the model control group,the Selenium Yeast Tablet group,and the Guben Tongjing group were significantly increased,and the difference was statistically significant(P <0.05);compared with the model control group,the m RNA expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of rats in the Selenium Yeast Tablet group and Guben Tongjing group were decreased,and the difference was statistically significant(P<0.05);compared with the Selenium Yeast Tablets group,the m RNA expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of rats in the Guben Tongjing group were decreased,and the difference was statistically significant(P<0.05).(6)Western blot results: compared with the blank control group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of the rats in the model control group,the Selenium Yeast Tablet group,and the Guben Tongjing group were significantly increased,and the difference was statistically significant(P<0.05);compared with the model control group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of the rats in the Selenium Yeast Tablet group and the Guben Tongjing group were significantly lower,and the difference was statistically significant(P<0.05);compared with the Selenium Yeast Tablet group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of the rats in the Guben Tongjing group were decreased,and the difference was statistically significant(P<0.05).(7)Immunohistochemistry results: the positive expression of TLR4 in the thyroid tissue of the rat showed light yellow,and it was mainly expressed in the cytoplasm of the thyroid follicular epithelial cells;the positive expression of My D88 appeared light yellow or brownish yellow,and it was mainly expressed in the cytoplasm of the thyroid follicular epithelial cells;the positive expression of NF-κB p65 was brownish yellow,and it could be found in the cytoplasm or nucleus of the thyroid follicular epithelial cells.Compared with the blank control group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of the rats in the model control group,the Selenium Yeast Tablet group and the Guben Tongjing group were significantly increased,and the difference was statistically significant(P<0.05);compared with the control group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of the rats in the Selenium Yeast Tablets group and the Guben Tongjing group were decreased,and the difference was statistically significant(P<0.05);compared with the Selenium Yeast Tablets group,the protein expression levels of TLR4,My D88,and NF-κB p65 in the thyroid tissue of rats in the Guben Tongjing group were decreased,and the difference was statistically significant(P<0.05).Conclusion1.The treatment method of Tuina combined with acupuncture under the guidance of Guben Tongjing theory can improve the clinical symptoms of patients,reduce the concentration of thyroid autoimmune antibodies,and reduce inflammation.It shows certain effectiveness and safety.Moreover,it enjoys certain advantages in early prevention,early treatment,and prevention of recurrence.Therefore,it is worthy of clinical promotion and application.2.Factors such as mechanical stimulation of changes in the physiological curvature of the cervical spine,thermal stimulation of local temperature changes in the thyroid gland,and chemical stimulation of emotional changes are related to the production of thyroid autoantibodies and the secretion of related hormones,which may be the potential cause of HT onset.3.The treatment method of Tuina combined with acupuncture under the guidance of Guben Tongjing theory can inhibit the expression of IL-6 and IL-12 and reduce inflammation by lowering down the protein expression levels of TLR4/My D88/NF-κB signaling pathway and m RNA,reduce inflammatory damage of the thyroid follicular structure,and protect the thyroid tissue. |
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