[Mechanism of modified Shaofu Zhuyu Decoction in antagonizing endometriosis angiogenesis based on mitophagy mediated by CaMKKβ/AMPK/HIF-1α signaling pathway]

This paper aims to elucidate the specific action mechanism of modified Jiawei Shaofu Zhuyu Decoction in antagonizing angiogenesis in endometriosis(EM) by regulating the Ca~(2+)/calmodulin-dependent protein kinase-2(CaMKKβ)/AMP-activated protein kinase(AMPK)/hypoxia-inducible factor 1α(HIF-1α) signaling pathway and inhibiting mitophagy. With the help of the random number table, the rats in the sham operation group was randomly selected from female non-pregnant SPF rats, and the remaining rats were established as EM models with kidney-Yang deficiency and blood stasis syndrome using the freeze-operation-freeze method. After successful modeling, the rats were randomly divided into a dienogest group, a high-dose modified Shaofu Zhuyu Decoction group, a low-dose modified Shaofu Zhuyu Decoction group, and a group of high-dose modified Shaofu Zhuyu Decoction + CaMKKβ inhibitor. The improvement of ectopic lesion volume was compared by observation and measurement, and the pathological changes of each group were evaluated by hematoxylin-eosin(HE) staining. The mitochondrial ultrastructure was observed by a transmission electron microscope. The level of mitochondrial reactive oxygen species(mtROS) was detected by MitoSOX Red fluorescent probe. The terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) method was used to detect the apoptosis of each group. The localization of CaMKKβ, AMPK, and HIF-1α in ectopic tissue was observed by immunohistochemistry. The protein and mRNA expressions of the CaMKKβ/AMPK/HIF-1α signaling pathway key proteins(CaMKKβ, AMPK, p-AMPK, and HIF-1α), mitophagy-related molecules(B-cell lymphoma-2(Bcl-2), Bcl-2/adenovirus E1B 19 kD interacting protein 3(BNIP3), microtubule-associated protein 1 light chain 3B(LC3B)) in ectopic tissues were detected by Western blot(WB) and reverse transcription-quantitative polymerase chain reaction(RT-qPCR). The expression levels of angiopoietin-2(Ang-2), vascular endothelial growth factor(VEGF), intercellular adhesion molecule-1(ICAM-1), and other angiogenesis-related factors in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA). Compared with those in the sham operation group, the rats in the model group exhibited significantly increased ectopic lesions. HE staining reveals vascular dilatation and congestion in the ectopic lesion, as well as interstitial hyperplasia with inflammatory cell infiltration. The mitochondrial swelling and cristae structure destruction were observed using the transmission electron microscope. MitoSOX Red fluorescent probe shows increased mtROS. Immunohistochemistry and WB results show that the CaMKKβ/AMPK/HIF-1α signaling pathway is abnormally activated, accompanied by excessive enhancement of mitophagy(increased expression of BNIP3, LC3B, and Bcl-2). After the intervention of dienogest, high-and low-dose modified Shaofu Zhuyu Decoction, and modified Shaofu Zhuyu Decoction combined with CaMKKβ inhibitor, the ectopic lesion volume of rats in each group was significantly reduced. HE staining was improved to varying degrees. The mitochondrial structure was gradually optimized as observed under a transmission electron microscope. At the same time, compared with those in the model group, the mtROS in the ectopic lesion of rats in each intervention group was significantly down-regulated. Immunohistochemistry, WB, and qPCR detection results show that the protein and mRNA expressions of key molecules of the signaling pathway(CaMKKβ, AMPK, p-AMPK, and HIF-1α) and mitophagy(BNIP3, Bcl-2, and LC3B) are significantly inhibited. The results of ELISA show that the expression of Ang-2, VEGF, and ICAM-1 in the serum of rats in each intervention group is significantly decreased, and the effect of modified Shaofu Zhuyu Decoction combined with the CaMKKβ inhibitor is significantly better than that of each intervention group. Modified Shaofu Zhuyu Decoction can promote the release of apoptotic factors by antagonizing mitophagy mediated by CaMKKβ/AMPK/HIF-1α signaling pathway and then antagonize the progress of EMs angiogenesis.