| Purpose:By observing the intervention effect of xiaoyin granule on thyroid autoantibodies,morphological structure and thyroid function in rats with EAT liver depression and spleen deficiency syndrome,the pathogenesis essence of hashimoto thyroiditis liver depression and spleen deficiency syndrome was expounded.And explore the anti-inflammatory and anti-apoptotic effect of Xiaoying granules on rats with eating liver depression and spleen deficiency by regulating the activation of the thyroid TLRs/MyD88/NF-?B signaling pathway and the balance of Treg/Th17 cells..Materials and methods: Firstly,the animal experiment was used to observe the intervention effect of Xiaoying granule on rats with EAT liver depression and spleen deficiency syndrome,and to explore the partial mechanism of action.After one week of adaptive feeding,48 SPF female SD rats were divided into two groups by random number method: normal group(12)and model group(36).Then,36 rats in the model group were subcutaneously injected with high-iodine water combined with thyroglobulin to induce hashimoto thyroiditis.By the 5th week of the experiment,the rats in the model group increased chronic bondage stress,excessive fatigue,dietary loss and other complex intervention factors,so as to replicate the liver depression and spleen deficiency syndrome.By the end of the eighth week of the experiment,hashimoto's thyroiditis model was evaluated by detecting the content of thyroid autoantibodies TGAb and TPOAb in serum.Weight of rats was weighed before and after modeling,and weight growth rate of each group was compared.At the 8th weekend of the experiment,the rats' sugar water preference experiment was conducted to observe the performance of depression and evaluate the model of liver depression and spleen deficiency.The rats successfully evaluated by the model were randomly divided into three groups again: model group(12 rats),Tripterygium wilfordii group(11 rats),and Xiaoying granule group(11 rats).Different drug interventions were given for 8 weeks.Rat dose was calculated by equivalent dose conversion method of body surface area between human and rat.Rats in the normal group and the model group were given 2ml distilled water twice a day for 8 weeks.Rats in the Tripterygium wilfordii group were given Tripterygium wilfordii polyglycoside tablets at the dose of 9.45mg/kg·d-1 through oral gavage for 8 weeks.Rats in the xiaoying granule group were given Xiaoying granule by oral gavage at the dosage of 16.17g/kg·d-1 for 8 weeks.After 8 weeks of treatment,blood of abdominal aorta and thyroid tissue of rats were taken to detect relevant indicators.The levels of TGAb,TPOAb,FT3,FT4,TSH,IL-10,IL-17 and IL-23 in serum of rats in each group were detected by enzyme-linked immunosorbent assay(ELISA);HE staining was used to detect the pathological morphological changes of thyroid tissue.The ultrastructure of thyroid gland was observed by electron microscope.TUNEL assay was used to detect the apoptosis of thyroid cells.The expressions of Foxp3?ROR?t?TLR2?TLR4?MyD88?NF-?Bp65 were detected by Western blot.Then,through the ancient literature mining and modern literature retrieval of "Yingbing" in traditional Chinese medicine,the pathogenesis of hashimoto thyroiditis and liver depression and spleen deficiency syndrome in traditional Chinese medicine and modern medicine were discussed,providing scientific theoretical support of traditional Chinese medicine and modern medicine for the treatment of hashimoto thyroiditis by soothing the liver and strengthening the spleen.Result: 1.Model evaluation: compared with the normal group,serum TGAb and TPOAb levels of thyroid antibodies in the model group were significantly increased,with statistically significant differences(P<0.01).Compared with the normal group,the glucose water consumption rate and body weight growth rate of rats in the model group were significantly reduced,with statistically significant differences(P < 0.05).2.General status: no abnormal appearance or behavior was observed in the normal group.Rats in the modeling group showed irritability at the early stage of modeling,strong resistance and struggle when shackled,and quickly escape and erect back hair after removing shackles.In the later stage of modeling,the rats in the modeling group gradually showed depression from irritability,decreased activities,liked to cluster together and arch back,weakened response to bondage,rough and fluffy back hair easy to fall off,dark yellow and lack of lustre,lethargy,tiredness,little food,and sometimes loose stools.After treatment,the above general conditions of rats in Tripterygium wilfordii group and Xiaoying granule group were significantly improved.Two unmoulded rats were removed,so the mould-forming rate was 94.44%.After treatment,a rat was taken from each group and pre-tested before the detection indicators.1 rat in the model group died due to improper gavage operation,and 1 rat in the normal group was hemolytic,so 10 complete samples of rats in each group were tested for the corresponding indicators.3.Serum TGAb and TPOAb levels in all groups of rats: compared with the normal group,serum TGAb and TPOAb levels in model group,tripterygium wilfordii group and xiaoying granule group were all increased,except for serum TPOAb levels in Xiaoying granule group,the difference was statistically significant(P< 0.01 or 0.05).Compared with model group,serum TGAb and TPOAb levels of rats in tripterygium wilfordii group and Xiaoying granule group were decreased,and the difference was statistically significant(P< 0.01).Compared with tripterygium wilfordii group,serum TGAb and TPOAb levels in Xiaoying granule group were decreased,and the difference was statistically significant(P < 0.01).4.Serum levels of FT3,FT4 and TSH in each group of rats:Compared with the normal group,serum FT3 and FT4 levels in the model group,Tripterygium wilfordii group and Xiaoying granule group were all decreased(P< 0.01 or 0.05),and TSH levels were increased(P< 0.01),with statistically significant differences.Compared with the model group,serum FT3 and FT4 levels in the Tripterygium wilfordii group and Xiaoying granule group were increased,while TSH levels were decreased,with statistically significant differences(P<0.01).Compared with Tripterygium wilfordii group,serum FT3 and FT4 levels in Xiaoying granule group were increased,and TSH levels were decreased,with statistically significant differences(P < 0.01).5.Pathological morphological changes of thyroid tissues in each group of rats:The thyroid gland in the normal group had complete structure and regular morphology,uniform follicular size and morphology,and no obvious lymphocyte infiltration in the stroma.In the model group,the thyroid tissue structure was damaged,some follicles were enlarged,and some follicles were atrophic.A large number of lymphocyte infiltration was observed in the follicular cavity and interstitial tissue,accompanied by interstitial fibrosis.In the Tripterygium wilfordii group,some thyroid follicles were enlarged,some follicular atrophy,lymphocyte infiltration in the follicular cavity and interstitial tissue was reduced,and the degree of fibrosis was reduced.The pathological morphological characteristics of thyroid gland in Xiaoying granule group were similar to those in tripterygium wilfordii group,and the number of lymphocyte infiltration in follicular cavity and interstitium was reduced without obvious fibrosis.6.Ultrastructural changes of thyroid gland under electron microscope: in the normal group,the cell size was moderate,the cell membrane was regular,the free surface microvilli were sparse,the nucleus was round,the nuclear membrane was clear and complete,the cytoplasm was uniform,there were abundant organelles,the rough endoplasmic reticulum was clear and distinct,and no apoptotic bodies were observed.In the model group,the cell body was wrinkled,the cell membrane was irregular,the free surface microvilli were increased and irregular,the nucleus was pyretic,the nuclear membrane was irregular or ruptured,the intracytoplasmic organelles were reduced,the endoplasmic reticulum was highly expanded and edema was observed,mitochondria were significantly reduced,and some mitochondrial cristae disappeared,showing the formation of apoptotic corpuscles.The cell body of Tripterygium wilfordii group was swollen,the cell membrane was relatively regular,the free surface microvilli were relatively regular,the nucleus was elliptic,the nuclear membrane was relatively thick and complete and clear,vacuoles of different sizes appeared in the cytoplasm,some organelles were dissolved,the endoplasmic reticulum was significantly swollen,and no apoptotic bodies were observed.In the Xiaoying granule group,the cell body was slightly swollen,the cell membrane was relatively regular,the free surface microvilli were few and regular,the nucleus was round or oval,the nuclear membrane was complete,the intracellular organelles were slightly reduced,a few vacuoles were observed,and the endoplasmic reticulum was slightly expanded.7.Apoptosis number of thyroid tissue cells in each group of rats:Compared with the normal group,the apoptosis of thyroid cells in model group,tripterygium wilfordii group and Xiaoying granule group increased,and the difference was statistically significant(P< 0.01).Compared with the model group,the apoptosis of thyroid cells in the tripterygium wilfordii group and Xiaoying granule group decreased,and the difference was statistically significant(P< 0.01).Compared with Tripterygium wilfordii group,the apoptosis of thyroid cells in Xiaoying granule group was slightly increased,but there was no statistical significance between the two groups.8.The expression levels of TLR2?TLR4?MyD88?NF-?Bp65 in thyroid tissue of rats in each group:Compared with the normal group,the expression levels of TLR2?TLR4?MyD88?NF-?Bp65 protein in thyroid tissue of rats in model group,Tripterygium wilfordii group and Xiaoying granule group increased significantly(P<0.01).Compared with the model group,the expressions ofTLR2?TLR4?MyD88?NF-?Bp65 were decreased in the thyroid tissues of rats in the Tripterygium wilfordii group and Xiaoying granule group,with statistically significant differences(P<0.01).Compared with Tripterygium wilfordii group,the expressions of TLR2?TLR4?MyD88?NF-?Bp65were increased in the thyroid tissues of rats with Xiaoying granules group,but there was no statistical significance.9.Serum IL-10?IL-17?IL-23 levels in all groups of rats: compared with the normal group,serum IL-10 levels in the model group,tripterygium wilfordii group and Xiaoying granule group were all decreased,while IL-17 and IL-23 levels were significantly increased,with statistically significant differences(P<0.01).Compared with the model group,serum il-10 levels in the Tripterygium wilfordii group and Xiaoying granule group were increased(P< 0.01 or 0.05),while IL-17 and IL-23 levels were decreased(P<0.01),with statistically significant differences.Compared with tripterygium wilfordii group,serum IL-10 levels in Xiaoying granule group were increased(P< 0.01),and IL-17 and IL-23 levels were decreased(P< 0.01),the differences were statistically significant.10.Foxp3?ROR?t protein expression in thyroid tissues of rats in each group: compared with the normal group,Foxp3 protein expression in thyroid tissues of rats in model group,tripterygium wilfordii group and Xiaoying granule group was decreased,while ROR?t protein expression was increased,with statistically significant differences(P< 0.01).Compared with the model group,the Foxp3 protein expression in thyroid tissues of rats in the tripterygium wilfordii group and Xiaoying granule group was increased,and the ROR?t protein expression was decreased,with statistically significant differences(P<0.01).Compared with tripterygium wilfordii group,Foxp3 protein expression increased and ROR?t protein expression decreased in thyroid tissues of rats in Xiaoying granule group,but there was no statistical significance.Conclusion: 1.After subcutaneous injection of high-iodine water combined with thyroglobulin to induce hashimotos thyroiditis in rats,combined with chronic bondage,excessive fatigue,dietary loss and other complex intervention factors,the model of eating liver depression and spleen deficiency combined with rats can be successfully established,which can better simulate the disease characteristics of clinical hashimotos thyroiditis liver depression and spleen deficiency.2.Xiaoying granule has a therapeutic effect on rats with eating liver stagnation and spleen deficiency by reducing thyroid autoantibodies,reducing thyroid tissue pathology and ultrastructural damage,and reducing hypothyroidism.3.By inhibiting the abnormal activation of the TLRs/MyD88/NF-?B signaling pathway,Xiaoying granules reduced the apoptosis of thyroid cells in rats with eating liver depression and spleen deficiency.It increases serum il-10 level and decreases serum il-17 and il-23 level,which plays an anti-inflammatory role.4.Xiaoying granules also play a role in inhibiting the inflammatory response of hashimotos thyroiditis model rats by regulating the disturbance of Treg/Th17 balance. |
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