Effects of Jianpi Bushen Formula （健脾补肾方） on the Autophagosomes and PI3K/Akt/mTOR Pathway in Radiation Injured Model Mice / 中医杂志

ABSTRACT

ObjectiveTo explore the effects and possible mechanisms of Jianpi Bushen Formula （健脾补肾方） on radiation-induced immune function damage of mice. MethodsFifty mice were randomly divided into four groups： normal group， model group， thymosin group， high- and low-dose groups of Jianpi Bushen Formula， with 10 mice in each group. Except for the normal group， the mice in the other groups were irradiated with a single whole-body dose of 6.0 Gy X-rays to establish a radiation-injured mouse model. After the successful modeling， the low- and high-dose groups of the Jianpi Bushen Formula were given respectively 13 g/（kg·d）、 26 g/（kg·d） of the formula by gavage， while the thymosin group was given 11.7 mg/（kg·d） of thymosin by gavage， and the normal group and model group were given 0.1 ml/（10g·d） of 0.9% sodium chloride solution by gavage. Each group was administered once a day for 7 consecutive days. After the last gavage， the mice were weighed， and their spleens were separated and weighed to calculate the spleen index. The levels of interferon gamma （IFN-γ） and interleukin 2 （IL-2） in the spleen tissue were detected by enzyme linked immunosorbent assay （ELISA）. The autophagosomes in the spleen were observed by transmission electron microscopy. The mRNA expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， and mammalian target of rapamycin （mTOR） in the spleen were detected by real-time fluorescent quantitative PCR. The protein expression of PI3K， Akt， and mTOR in the spleen， as well as the expression of autophagy-related proteins microtubule-associated light chain protein 3 （LC3）， Beclin1， and p62 were detected by Western blot. ResultsCompared with the normal group， the model group showed significant decreases in body weight， spleen index， and levels of IFN-γ and IL-2 in the spleen （P＜0.01）； the mRNA and protein expression levels of PI3K， Akt， and mTOR in the spleen were also significantly reduced （P＜0.01）； the expression of Beclin1 protein， the ratio of LC3-II/LC3-I significantly increased （P＜0.01）， while the level of p62 protein expression significantly decreased （P＜0.01）. And transmission electron microscopy showed a significant increase in the number of autophagosomes in the spleen and severe cell structure damage in the model group. Compared with the model group， all the above indicators in each medication group were significantly improved （P＜0.05 or P＜0.01）. In the high-dose Jianpi Bushen Formula group， partial intact cristae were visible in the fine mitochondria of the spleen， and there were more autophagosomes. In the low-dose Jianpi Bushen Formula group and thymosin group， the structure of the fine mitochondria in the spleen was relatively intact， and there were fewer autophagosomes. The improvement effect of the low-dose Jianpi Bushen Formula group was better than that of the high-dose group （P＜0.05 or P＜0.01）， and there was no significant difference between the low-dose group and the thymosin group in terms of each indicator （P＞0.05）. ConclusionJianpi Bushen Formula may alleviate the structural damage of the spleen， promote the recovery of immune function， and achieve a best effect at a low dose by enhancing the PI3K/Akt/mTOR signaling pathway in the spleen and inhibiting the over-activation of autophagy induced by radiation.