Study On The Mechanism Of Zhenwu Decoction In Treating Kidney-Yang Deficiency Hypothyroidism Based On Keap1/Nrf2 Pathway

Objective:To observe the therapeutic effect of Zhenwu Decoction on hypothyroidism model rats,and to explore its mechanism based on Keap1/Nrf2 signaling pathway,so as to provide experimental basis for the clinical application of Zhenwu Decoction in the treatment of hypothyroidism.Methods:Rats were given 0.1%PTU at a dose of 10 mg·kg^-1·d^-1for 15 days to establish a hypothyroidism model and evaluate the model.The rats with successful model evaluation were randomly divided into model group,Zhenwu Decoction low dose group,Zhenwu Decoction middle dose group,Zhenwu Decoction high dose group and positive drug levothyroxine sodium（L-T₄）group（n=11）,and normal control group（n=10）.The intragastric doses of low,medium and high dose groups of Zhenwu Decoction were 1.89/3.78/7.56 g·kg^-1·d^-1,and the intragastric dose of L-T₄group was 9μg·kg^-1·d^-1.After 28 days of administration,the whole state of each group was observed.The levels of FT₄,FT₃,TSH,E₂,T and CRH in serum were detected by ELISA.HE staining technique was used to detect the pathological changes of thyroid tissue,and the intervention effect of Zhenwu Decoction on hypothyroidism model rats was observed.The contents of MDA,CAT and SOD in serum and thyroid tissue of rats in each group were detected by TBA,ammonium molybdate and WST-1 method.Western Blot and Q-PCR technique were used to detect the protein and mRNA expression of Nrf2,Keap1and Tg in thyroid tissue,and to explore the mechanism of Zhenwu Decoction on hypothyroidism model rats.Results:1.Compared with the normal control group,the body weight,anal temperature,food intake and water intake of the rats in the model group were significantly decreased（P<0.01）,the urine output was significantly increased（P<0.05）,the FT₄level was significantly decreased（P<0.05）,and the TSH level was significantly increased（P<0.01）,which was in line with the evaluation criteria of hypothyroidism model.The success rate of modeling was 91.7%.2.Compared with the normal control group,the body weight,anal temperature,food intake and water intake of the model group were significantly decreased,and the urine output was significantly increased（P<0.01）.The levels of FT₄,FT₃,E₂,T and CRH were significantly decreased,and the level of TSH was significantly increased（P<0.01）.Compared with the model group,the body weight,food intake and water intake of rats in the middle dose group of Zhenwu Decoction increased significantly（P<0.05）.The rectal temperature of rats in each treatment group was significantly increased,the urine volume was significantly decreased（P<0.01）,and the levels of FT₄,FT₃,E₂,T and CRH were significantly increased（P<0.01）.3.HE results showed that the follicles in the thyroid tissue of the model group decreased,the colloid in the follicular cavity decreased or disappeared,and there were congestion and bleeding changes between the follicles.Compared with the model group,the thyroid tissues of rats in each treatment group showed increased follicles,glial formation,increased small cell follicles in focal glands,and a small amount of congestion and bleeding in the stroma.4.The results of MDA,CAT and SOD showed that compared with the normal control group,the content of MDA in serum and thyroid tissue of the model group increased significantly,and the content of CAT and SOD increased significantly（P<0.01）.Compared with the model group,MDA in serum and thyroid tissue of rats in Zhenwu Decoction high dose group and L-T₄group decreased significantly,and SOD increased significantly（P<0.05）.The content of CAT in the middle dose group of Zhenwu Decoction was significantly increased（P<0.05）,and that in the L-T₄group was significantly increased（P<0.01）.5.Western Blot results showed that compared with the normal control group,the expression of Nrf2 and Tg in the model group was significantly decreased（P<0.01）,and the expression of Keap1 was significantly decreased（P<0.05）.Compared with the model group,the expression of Nrf2 in the thyroid tissue of rats in the low-dose Zhenwu Decoction group and the L-T₄group increased（P<0.05）;the expression of Keap1 in the Zhenwu decoction dose group was significantly increased（P<0.05）.The expression of Tg in each treatment group increased（P<0.01）.6.Q-PCR results showed that compared with the normal control group,the mRNA expression of Nrf2,Keap1 and Tg in the model group was significantly decreased（P<0.01）.Compared with the model group,the mRNA expression of Nrf2 and Keap1 in the high dose group of Zhenwu Decoction increased significantly.The mRNA expression of Tg in Zhenwu Decoction low dose group was significantly increased（P<0.05）,and that in Zhenwu Decoction medium dose group was significantly increased（P<0.01）.The mRNA expression of Nrf2 and Tg in the L-T4 group was significantly increased（P<0.01）,and the mRNA expression of Keap1 was significantly increased（P<0.05）.Conclusion:1.Hypothyroidism model can be successfully established by PTU gavage for 15 days.2.Zhenwu Decoction can significantly improve the overall state and thyroid function of hypothyroidism model rats,regulate the function of hypothalamus-adrenal gland and gonadal axis,reduce thyroid tissue damage,and have a therapeutic effect on hypothyroidism model rats.3.Zhenwu decoction can activate Keap1/Nrf2 signaling pathway,up-regulate the expression of Nrf2,Keap1 and Tg,increase the content of antioxidant enzymes SOD and CAT,reduce the content of peroxide MDA,and then reduce the oxidative stress level of hypothyroidism model rats,which is confirmed to be one of the mechanisms of Zhenwu decoction in the treatment of hypothyroidism.