Effects Of Gingko Biloba Extract On Aortic Calcification In Chronic Renal Failure Rats Induced By High Phosphorus

ObjectiveCardiovascular disease (CVD) is one of the main complications and causes of death in patients with chronic kidney disease (CKD). Vascular calcification is ubiquitous in patients with CKD, and there is still no better clinical way for prevention of vascular calcification, so elucidate its mechanism and find the effective therapeutic measure is important to prevent the CVD in patients with CKD. In recent years, studies have shown that vascular calcification in CKD were caused by unbalanced promoting and inhibiting factors, and its mechanism is complex. epidemiological and clinical research data show that hyperphosphatemia is one of the major risk factors of inducing vascular calcification in patients with CKD.Recently, herbal therapy has been gaining popularity among clinicians due to their beneficial effects with minimum toxicity. The Ginkgo biloba extract (EGb 761) is a standardized extracts of dried leaves and it can stabilize the changes in mitochondrial membrane potential occurred due to excessive ROS, help preventing the disruption of ionic homeostasis and repressing atherosclerosis. However, the pharmacological potential and mechanism of action of EGb 761 in rats vascular smooth muscle cells (VSMCs) calcification or vascular calcification in patients with CKD is yet to be explored.High phosphorus could induce VSMCs transdifferentiate into osteoblast-like cells and promote vascular calcification through increasing mitochondrial ROS production, and EGb 761 has antioxidant activity and reduce mitochondrial ROS generation, so we speculate that EGb 761 might inhibite calcification of vascular smooth muscle cells and prevent the vascular calcification in patients with CKD by reducing the generation of mitochondrial ROS.Materials and MethodsTo find out the effects of EGb 761 on vascular calcification with chronic kidney disease, this study is divided into in vitro cell experiments and in vivo animal experiments.The in vitro cell experiments are carried out by using high phosphorus induce VSMCs transdifferentiate into osteoblast-like cells and testing related pathway signals and cellular calcium content, observing effects of β-glycerophosphate and EGb 761 on vascular smooth muscle cells calcification. In vivo animal models of high phosphorus-induced aortic calcification in chronic renal failure rats were fed with or without EGb 761, and its effects on animals calcification were studied to elucidate the molecular mechanism.1. rat aortic vascular smooth muscle cell were cultured by explant culture method and identificated, and intervented with β-glycerophosphate or different concentrations of EGb 761 for 7 days.2. Calcium content in the cells, alkaline phosphatase (ALP) activity, cell protein content, NF-κB activation and reactive oxygen species (ROS) production were assayed respectively, and the expression of core binding factor-α1 and a-SM actin mRNAwas detected by RT-PCR.3. Use high phosphorus induced rats with chronic kidney disease to establish the vascular calcification model with or without the intervention of EGb 761 for 16 weeks, detect the serum urea nitrogen, creatinine, calcium, phosphorus of the rats in group respectively.4. The aortic calcium content of the whole section of the abdominal aorta vessel wall was detected by Cresolphthalein complexation colorimetry. Vascular calcification of the upper thoracic aorta vessel was detected by von kossa calcification staining in each group. Protein expression of NF-κB p65, core binding factor-a1 (cbfa1) and a-SM actin of the thoracic aorta vessel wall were detected by Western blotting analysis.Results1. The calcium contents of the BGP group were significantly higher than those of the control group (P<0.01), and were inhibited by EGb 761 in a concentration-dependent manner (P<0.05).2. Data showed β-glycerophosphate induced the enhanced expression of ALP, up-regulated the NF-κB activity and increased ROS production of VSMCs while these decreased when administrated with EGb 761 (P<0.05), and EGb 761 inhibited (3-glycerophosphate induced expression of cbfa1, upregulated a-SM actin mRNA gene expression in VSMC.3. Biochemical detections showed that blood urea nitrogen, creatinine, serum phosphorus of high phosphorus induced aortic calcification in chronic renal failure group (HPIAC group) and high phosphorus induced aortic calcification in chronic renal failure with EGb 761 interfered group (HPIAC+EGb 761 group) were significantly higher than the sham operated control group (P<0.05), and there were no significant differences between the HPIAC group and HPIAC+EGb 761 group(P>0.05).4. Compared with the sham operated control group, the aortic calcium contents in HPIAC group and HPIAC+EGb 761 group were significantly higher (P<0.05), and that in HPIAC+EGb 761 group was lower than the HPIAC group (P<0.05).5. By Western blotting analysis, Protein expressions of NF-κB p65 and core binding factor-a1 (cbfa1) in HPIAC groups were detected significantly higher than the sham operated control group(P<0.05), and significantly decreased in the HPIAC+EGb 761 group(P<0.05). And Protein expression of a-SM actin in HPIAC group were detected significantly lower than the sham operated control group (P<0.05), and significantly increased in the HPIAC+EGb 761 group(P<0.05).Conclusion1. EGb 761 significantly reduced calcification induced by β-glycerophosphate in rat aortic vascular smooth muscle cells. It not only reduced the deposition of calcium, but also inhibited osteogenic transdifferentiation, which may be associated with decreased expression of ALP, down-regulated the NF-κB activity and reduced ROS production of VSMCs, and may have the potential to serve as a role for vascular calcification in clinical situations.2. EGb 761 can improve high phosphorus induced aortic calcification in chronic renal failure model of rat, and the mechanism might execute by ROS-NF-κB pathway, but the accurate pathway needs to be researched in depth.