| ObjectiveBased on the observation of symptoms, thyroid functions, thyroid tissue pathology on the animal model of Graves’disease, to evaluate the pharmacodynamic effect of JiaKangning capsule comprehensively. To provide a powerful laboratory evidence for clinical application, a reference for the popularization and application of "nourishing yin and suppressing hyperactive yang, reducing phlegm and resolving masses" treatment as well. To proceed with the TRAb mediated Akt signaling pathway, through the expression of the key protein Akt, mTOR, Bcl-2, Bax, cyclin D1, PCNA to investigate the effect of JiaKangning on Akt signaling pathway. Reveal the molecular mechanism of JiaKangning in the treatment of Graves’ disease.MethodsBuild animal models of Graves’ disease by immunizing BALB/c mice with Ad-TSHRa-289. GD mice were randomly divided into Normal control group, Ad-null control group, GD model control group, JiaKangning intervened group, Methimazole intervened group. Observe the mice activity status and body weight change during drug administration. After 5 weeks, the serum and thyroid tissue specimens were taken. To analyze the serum T4, T3, TSH levels in mice by radioimmunoassay, the serum TRAb level in mice by radioreceptor assay, make thyroid tissue HE staining. Thus make an integrated evaluation of JiaKangning’s effect. Using Western blot analysis detect the thyroid tissue Akt, p-Akt, mTOR, p-mTOR, Bcl-2, Bax, cyclin D1 and PCNA expressions, to find out the influence of JiaKangning’s effect on the signaling pathway.Results1 JiaKangning’s effect on symptoms and physical signs in GD miceTwo weeks after the drug intervention, weight in mice of JiaKangning and Methimazole intervened groups began to rebound. For 4 weeks, The weight of JiaKangning group (22.25±0.56) were higher than GD model control group (21.18±1.74), but no significant differences. The weight of Methimazole group (22.73±1.13) were higher than GD model control group significantly. There was no significant difference between the two above at the end of the dosing. In drug intervention phase, symptoms of hyperthyroidism compared with GD model control group, JiaKangning group and Methimazole group gradually improved over time.2 JiaKangning’s effect on thyroid functionJiaKangning group compared with itself before and after administration, TRAb levels decreased significantly (53.76±22.90vs16.90±20.75, p<0.01), T4 levels decreased significantly (204.03±52.22vs150.24±51.01, p<0.01). Five weeks after the treatment, TRAb, T4, T3 level of JiaKangning group compared with GD model control group were significantly lower (5.69±3.81vs41.61±28.63; 64.21±19.53vsl22.18±16.63; 0.98±0.57vs3.07±0.64, p<0.01) and showed no significant differences with Methimazole group. But there was no significant differences between groups in TSH level.3 JiaKangning’s effect on thyroid tissue morphologyAfter JiaKangning intervention, thyroid color, size, organizational structure of GD mice have a certain degree of recovery than GD model control group, Thyroid gross observation results were consistent with the HE staining.4 JiaKangning’s effect on p-Akt, p-mTOR, Bcl-2, Bax, cyclin D1, PCNA expressionAfter drug intervention, p-Akt/β-actin and p-Akt/Akt relative expression of JiaKangning group were lower than GD model control group showed no significant differences (0.29±0.05vs0.33±10.06; 0.57±0.10vs0.66±0.10, p>0.05). While Methimazole group were significantly lower than those in GD model group (0.27±0.04vs0.33±0.06; 0.53±0.07vs0.66±0.10, p<0.05). p-mTOR/β-actin and p-mTOR/mTOR relative expression of JiaKangning group were significantly lower than GD model control group (0.36±0.05vs0.42 ±0.05; 0.69±0.08vs0.82±0.11, p<0.01). Methimazole group were significantly lower than those in GD model group (0.34±0.03vs0.42±0.05; 0.67±0.09vs0.82±0.11, p<0.01) as well. There was no significant differences between the two groups above (p>0.05). Bcl-2/β-actin relative expression of JiaKangning group was significantly lower than GD model control group (0.23±0.05vs0.31±.12, p<0.05). Methimazole group were significantly lower than those in GD model group (0.24±0.03vs0.31±0.12, p<0.05) as well. There was no significant differences between the two groups above (p>0.05). Bax/β-actin relative expression showed no significant differences between groups (p>0.05). Bcl-2/Bax relative expression of JiaKangning group was lower than GD model control group showed no significant differences (0.94±0.55vsl.17±0.41, p>0.05). While Methimazole group were significantly lower than those in GD model group (0.77± 0.15vs1.17±0.41, p<0.05). cyclin Dl/β-actin relative expression of both JiaKangning group and Methimazole group were lower than GD model control group showed no significant differences (0.28±0.08vs0.36±0.10; 0.31±0.09vs0.36±0.10, p>0.05). PCNA/β-actin relative expression of JiaKangning group was significantly lower than GD model control group (0.21±0.04vs0.28±0.07, p<0.05). While Methimazole group had no significant difference from GD model group (0.23±0.05vs0.28±0.07, p>0.05).Conclusion1 With the Ad-TSHRa-289 immunized BALB/c female mice can be successfully induced animal models of Graves’ disease, which has clinical feature similarity, high rate of successful modeling and Stability.2 JiaKangning can effectively relieve hyperthyroidism of GD mice. It regulates the immunologic function and reduce pathogenic antibody TRAb. It repairs the thyroid pathological changes in GD mice.3 JiaKangning has effect on Akt signaling pathway. It can decrease the expression of Akt, mTOR, cyclin D1 and PCNA, which may be one of the mechanism of JiaKangning in inhibiting thyroid hormone synthesis and secretion as well as the thyroid cell proliferation.4 JiaKangning has effect on Akt signaling pathway. It can adjust the expression of Bcl-2 and Bax, which may be one of the mechanism of JiaKangning in inducing thyroid cell apoptosis.
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