ObjectiveTo investigate the in vitro effect of pseudolaric acid B (PAB) on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo, as well as its possible mechanisms, in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis. MethodsIn vitro experiments: the protoscoleces, vesicles, germinal cells, human foreskin fibroblasts (HFFs) and normal human liver cells were treated with different concentrations of PAB (0, 2.5, 5, 10, 20, 40, 80, 160 and 320 μmol/L) for 7, 5, 5, 5 and 5 days, then evaluated the survival rate of the protoscoleces, the release level of phosphoglucose isomerase (PGI) from the vesicles, the viability of the germinal cells, as well as the viability of HFFs and normal human liver cells. The protoscoleces and vesicles were fixed with 2.5% glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation. Animal experiments: the protoscoleces were isolated from the abdominal lesions of the protected gerbils, and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models, dividing into 3 groups with 6 mice in each group. The model group was given 0.3 mL of PBS by gavage daily, the albendazole (ABZ) group was given 0.3 mL ABZ (100 mg/kg) daily by gavage, the PAB group was given 0.3 mL of PAB (40 mg/kg) by gavage daily. After continuous gavage for 6 weeks, the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and cysteinyl aspartate specific proteinase3 (caspase3), the expression levels of nitric oxide (NO) was detected using a biochemical detection kit, Western blot was used to detect the expression levels of BCL2-associated X protein (Bax), B-cell lymphoma-2 (Bcl2), caspase3, cleaved-caspase3, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), phosphatidylinositol 3 kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKI (p-AKT) protein. ResultsIn vitro experiments: the protoscoleces of Echinococcus multilocularis were cultured with different concentrations of PAB for 7 days in vitro, the protoscoleces of 40, 80, 160 and 320 μmol/L group all died after 6, 4, 2 and 1 day, respectively; PAB exhibited a certain time and concentration dependence on the protoscoleces of Echinococcus multilocularis. After PAB treatment, the release of PGI in culture supernatant of Echinococcus multilocularis gradually increased with the increase of PAB concentration [concentration for 50% of maximal effect value was (24.40±1.42) μmol/L], the vitality of germinal cells was significantly inhibited [half maximal inhibitory concentration value was (15.94±2.55) μmol/L]. PAB had no significant toxicity to mammalian cells. When 20 μmol/L PAB intervention in the protoscoleces for 3 days, the expression levels of Bax and caspase3 proteins were upregulated, while the expression level of Bcl2 protein was downregulated. Animal experiments: compared with the model group, the wet weight of lesions in the PAB and ABZ groups decreased (P<0.01), and the inhibition rates of lesion growth in the PAB and ABZ groups were 91.03% and 74.44%, respectively. The expression of proliferation and angiogenesis indicators (Ki67, CD34, VEGF, VEGFR2, eNOS, NO) were downregulated in the lesion host microenvironment tissues of mice in the ABZ and PAB groups (P<0.05), while the expression of apoptosis related proteins (caspase3, cleaved-caspase3 and Bax) were upregulated and the expression of PI3K/AKT signaling pathway related proteins (p-PI3K and p-AKT) were downregulated (P<0.05). ConclusionPAB has a strong in vitro and in vivo effect against Echinococcus multilocularis, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway, leading to increased apoptosis and decreased angiogenesis.
Objective To evaluate the effectiveness of biodegradable magnesium alloy materials in promoting tendon-bone healing during rotator cuff tear repair and to investigate their potential underlying biological mechanisms. Methods Forty-eight 8-week-old Sprague Dawley rats were taken and randomly divided into groups A, B, and C. Rotator cuff tear models were created and repaired using magnesium alloy sutures in group A and Vicryl Plus 4-0 absorbable sutures in group B, while only subcutaneous incisions and sutures were performed in group C. Organ samples of groups A and B were taken for HE staining at 1 and 2 weeks after operation to evaluate the safety of magnesium alloy, and specimens from the supraspinatus tendon and proximal humerus were harvested at 2, 4, 8, and 12 weeks after operation. The specimens were observed macroscopically at 4 and 12 weeks after operation. Biomechanical tests were performed at 4, 8, and 12 weeks to test the ultimate load and stiffness of the healing sites in groups A and B. At 2, 4, and 12 weeks, the specimens were subjected to the following tests: Micro-CT to evaluate the formation of bone tunnels in groups A and B, HE staining and Masson staining to observe the regeneration of fibrocartilage at the tendon-bone interface after decalcification and sectioning, and Goldner trichrome staining to evaluate the calcification. Immunohistochemical staining was performed to detect the expression of angiogenic factors, including vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2), as well as osteogenic factors at the tendon-bone interface. Additionally, immunofluorescence staining was used to examine the expression of arginase 1 and Integrin beta-2 to assess M1 and M2 macrophage polarization at the tendon-bone interface. The role of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in tendon-bone healing was further analyzed using real-time fluorescence quantitative PCR. Results Analysis of visceral sections revealed that magnesium ions released during the degradation of magnesium alloys did not cause significant toxic effects on internal organs such as the heart, liver, spleen, lungs, and kidneys, indicating good biosafety. Histological analysis further demonstrated that fibrocartilage regeneration at the tendon-bone interface in group A occurred earlier, and the amount of fibrocartilage was significantly greater compared to group B, suggesting a positive effect of magnesium alloy material on tendon-bone interface repair. Additionally, Micro-CT analysis revealed that bone tunnel formation occurred more rapidly in group A compared to group B, further supporting the beneficial effect of magnesium alloy on bone healing. Biomechanical testing showed that the ultimate loads in group A were consistently higher than in group B, at 4 weeks, the stiffness of group A was also greater than that of group B, indicating stronger tissue-carrying capacity following tendon-bone interface repair and highlighting the potential of magnesium alloy in enhancing tendon-bone healing. Immunohistochemical staining results indicated that the expression of VEGF and BMP-2 was significantly upregulated during the early stages of healing, suggesting that magnesium alloy effectively promoted angiogenesis and bone formation, thereby accelerating the tendon-bone healing process. Immunofluorescence staining further revealed that magnesium ions exerted significant anti-inflammatory effects by regulating macrophage polarization, promoting their shift toward the M2 phenotype. Real-time fluorescence quantitative PCR results demonstrated that magnesium ions could facilitate tendon-bone healing by modulating the PI3K/AKT signaling pathway, which represented one of the molecular mechanisms driving this healing process. ConclusionBiodegradable magnesium alloy material accelerated fibrocartilage regeneration and calcification at the tendon-bone interface in rat rotator cuff tear repair by regulating the PI3K/AKT signaling pathway, thereby significantly enhancing tendon-bone healing.