| BACK GROUND AND OBJECTIVE:Chronic kidney disease(Chronic kidney disease,CKD)is a global public health problem.Vascular calcification(Cardiovascular disease,VC)is the main pathological basis of chronic kidney disease complicated with cardiovascular disease(Cardiovascular disease,CVD)and high risk factor in chronic kidney disease.Calcium and Phosphorus metabolism disorders,uremia,micro inflammation and other internal environment caused by Chronic kidney disease will lead to a variety of mechanisms involved in the development of vascular calcification,including vascular smooth muscle cells(VSMCs)osteogenesis changes,,Apoptosis,etc.A variety of signal paths and factor control this process,but the pathogenesis of Vascular calcification in chronic kidney disease is complex,has not yet elucidated.Modern medicine for the treatment of CKD vascular calcification focused on the control of its risk factors,without effective way,and the therapeutic effect is single,so in the current treatment for Vascular calcification in chronic kidney disease vascular calcification is difficult and popular.Mentor put forward "Invigorating kidney and activating blood,Removing turbidity and removing blood stasis "treatment according to Chinese medicine practitioners and combined with clinical experience.A large number of clinical and preoperative studies have shown that Bushen Huoxue Decoction can protect renal function,and improve the overall quality of life in patients with chronic renal failure,also correct the disorder of calcium and phosphorus metabolism.It has been confirmed that Bushen Huoxue Decoction can inhibit aortic smooth muscle cell osteogenesis changes to inhibit aortic vascular calcification.However,the mechanism of Bushen Huoxue Decoction on calcification of chronic kidney disease is not fully elucidated.Based on the previous research results,this study deeply explored and studied the mechanism of prevention and treatment of vascular calcification in chronic kidney disease based on mechanisms of apoptosis and autophagy.Research methods:1.90 rats were randomly divided into normal group,model group,calcitriol group,high dose group of traditional Chinese medicine,middle dose group and low dose group of Chinese medicine group.In addition to the normal group rats,the rats of other groups were given 250mg/kg dose of adenine suspension in the first 1-4 weeks and were feed 1.2%high phosphorus.In 5-8th weeks 125mg/kg of adenine were given every other day and 1.2%high-phosphorus diet.At the same time,the traditional Chinese medicine groups were treated with Bushen Huoxue Decoction.The dose of traditional Chinese medicine was calculated according to the body surface area of rats(1:6.25)and 4 times,2 times and 1 time dose of clinical adult dosage,high dose group(5.6g/kg · day),middle dose group(2.8g/kg · day),low dose group(1.4g/kg · day).The rats in the normal group were given 0.9%sodium chloride solution for 8 weeks.At the end of the administration,the general performance of the rats was observed and the body weight was measured.The changes of kidney were observed and renal pathology changes were observed by HE staining and Masson staining.The calcification nodules of aorta were observed by alizarin red staining.The ultrastructural changes of autophagy and calcified nodules were observed by transmission electron microscopy.(Ca),phosphorus(Pi),serum creatinine(CREA)and urea nitrogen(UREA)were measured by abdominal aorta.The expressions of a-SMA,ALP,Bcl-2,Bax,Caspase-3,LC3-?/?,Beclinel,Akt,mTOR protein and phosphorylation were detected by Western blotting.2.The vascular smooth muscle cells were treated with different concentrations of phosphorus(Pi 1.4mmol/L,Pi 2.0mmol/L,Pi 2.6mmol/L,Pi 3.2mmol/L)in vitro to establish vascular smooth muscle cell calcification Model,to simulate the microenvironment of vascular calcification.The effects of different concentrations of phosphorus on the calcification of vascular smooth muscle cells were observed by alizarin red staining,calcium content detection and MTT assay at different time points(3d,6d,9d,12d).On the 9th day,the relationship between apoptosis and calcification of vascular smooth muscle cells was observed by flow cytometry.The changes of ROS content in vascular smooth muscle cells(VSMCs)were observed at different time points under the condition of Pi2.6mmol/L calcification model.At the same time,the relationship between apoptosis and vascular smooth muscle cell calcification was observed on the 9th day by alizarin red staining and cell calcium deposition at different concentrations of inhibitor of apoptosis(1uM,2.5uM,5uM,10uM).3.Sixty SD rats were randomly divided into two groups after being weighed.The rats in the blank group(n=15)were given normal saline.The rats in the other groups were treated with Bushen huoxue Decoction according to 3g/100gin the morning and evening for 5 times.The last gavage was done after 12h of fasting,and 1h later,the rats were given anesthesia,abdominal aorta blood collection,centrifugation,inactivation treatment to make serum and-80? preservation for later use.The method of culture in vitro and subculture was taken to establish the vascular smooth muscle cell calcification Model with Pi 2.6 mmol/1 mediun and the rats were treated with different concentrations of Bushen Huoxue Decoction(5%,10%and 20%).The degree of calcification of vascular smooth muscle cells was determined at different time by alizarin red staining and calcium content.On the 9th day the changes of blue fluorescence were observed by DAPI staining and flow cytometry was used to analyze the effect of Kidney-containing serum on apoptosis The changes of ROS content were observed on the 6th day by detecting the normal group,the model group and the serum-containing middle dose group.The expression of a-SMA,ALP,Bcl-2,Bax,Caspase-3,LC3-II/I,Becline 1,Akt and p-mTOR protein in vascular smooth muscle cells were detected by Western-Blot method.Research result:1.Animal manifestations:Compared with the normal group,the body volume of the rats in model group decreased significantly.The hair scattered.The weight growth was slow.The rats manifested polydipsia,polyuria.Body hair dried off.The case in Calcitriol group was as well.Compared with the model group,the hair color and mental state of the rats were better in the traditional Chinese medicine groups,especially in the middle dose and high dose.Kidney appearance and manifestations:The liver and kidney indexes of the model group were significantly increased(P<0.01).The difference between the medium dose and the high dose group is obvious(P<0.01).HE staining showed that the number of normal glomeruli in the traditional Chinese medicine group was more than that in the model group,the degree of renal tubular dilatation was decreased,the inflammatory infiltration decreased and the adenine crystallinity decreased.The blue collagen fibers of Masson sataining model group deposited obviously and the difference was statistically significant(P<0.01)in the blue-stained area.Compared with the model group,the collagen concentration in the traditional Chinese medicine groups was significantly lower(P<0.01),especially in middle and high dose groups(P<0.01).Changes of biochemical indexes:Compared with the normal group,the levels of P,CREA and UREA in the model group were significantly higher while the level of serum calcium decreased(P<0.01).Compared with the model group,the levels of P,CREA and UREA in the middle dose group decreased(P<0.05),the serum calcium increased(P<0.01)in the 4th week and the levels of P,CREA and UREA significantly decreases(P<0.01),while the serum calcium increased in the 8th week(P<0.01).In the 8th week,the serum P significantly decreased and the serum Ca increased in the calcitriol group(P<0.05).Vascular calcification:Alizarin red staining showed that calcification in Chinese medicine groups reduced and the reduction of middle dose and high dose groups was more obvious.Under the transmission electron microscope,the smooth muscle cells could be seen structural integrity in the normal group,and the organelles such as mitochondria and endoplasmic reticulum were normal without deposition.The organization structures in the model group and the calcitriol group were completely destroyed with a large number of black calcification crystals.The situation in Chinese medicine groups eased especially in the middle dose and high dose groups.In Calcification group smooth muscle cells were completely destroyed and heterochromatin increased.In the model group the more active autophagy process could be observed and autophagy bicyclic body could be seen with a large number of calcification.Western-Blot result:there was a significant increase of expression of aorta ALP protein,a significant decrease of expression of ?-SMA protein(P<0.01),a decrease of apoptosis inhibitory protein BCL-2,an up-regulation of the expression of Bax and Caspase-3(P<0.01),a significant decrease of the expression of p-mTOR protein(P<0.01),and an up-regulation of the expression of LC3 and Beclin 1 protein(P<0.01).Western blot results showed that there was a decrease of the expression of ALP,Bax and Caspase-3 and an increase of the expression of a-SMA and Bcl-2 protein in Chinese medicine middle dose and high dose groups(P<0.05,P<0.01).There was a significant decrease of the expression of LC3-? protein in the calcitriol group and traditional Chinese medicine groups(P<0.01,P<0.05).There was an increase of the expression of mTOR protein and a decrease of the Beclin 1 protein in the middle dose group and high dose group.2.The establishment and evaluation of calcification model of vascular smooth muscle cells induced by high phosphorus showed that the normal cells were normal,not colored or pale red under microscopic after alizarin red staining.In the high phosphorus model group,a lot of cells fell off,orange red particles formed,and gradually fused into pieces or nodules.The cell morphology changed in the circular or irregular shape.Cell shrinked.The vacuoles appeared in cytoplasm.Cell refraction decreased.Sometimes local large tracts of cells fell off,and died.With the increase in phosphorus concentration and the extension of time,the degree of calcification was higher.Calcium content showed a positive correlation with phosphorus concentration and time.Apoptosis inhibitors can inhibit calcification of smooth muscle cells in a concentration-dependent manner.Flow cytometry showed that the apoptotic rate increased with the number of days,and was positively correlated with calcification.ROS was the highest on 6th day and gradually weakened over time.3.The effect and mechanism of Bushen Huoxue Decoction on the calcification of vascular smooth muscle cells at the cellular level was studied.The results showed that the normal cells were normal,not colored or pale red under microscopy after alizarin red staining.In the high phosphorus model group a large number of cells fell off,and orange red particles formed,and gradually fused into flaky or nodules.In Chinese medicine containing serum dose groups and apoptosis inhibitor group the number of calcified nodules reduced,and had a concentration-dependent.DAPI staining showed that the apoptotic cells increased in the model group.In the serum dose groups and the inhibitor group,the blue fluorescence was relatively weak,and the middle dose and the high dose of the traditional Chinese medicine were higher.Flow cytometry showed that of the 9th day,the apoptotic rate in the model group was significantly higher(P<0.01,n=3).Compared with model group,in inhibitor group and traditional Chinese medicine group,the apoptotic rate significantly decreased(P<0.01,P<0.05)and the effect of Chinese medicine group was dose-dependent.Compared with the model group,the ROS content in traditional Chinese medicine groups significantly decreased,Western-bloting results showed that the serum could decrease the expression of ALP,Bax,Caspase-3,LC3-?/? and Becline 1 and could increase the expression of a-SMA and Bcl-2,and could activate the expression of Akt protein,mTOR Signal protein.Conclusion:1.Bushen Huoxue Decoction can reduce the level of blood phosphorus in rats with chronic kidney disease,increase blood calcium,correct calcium and phosphorus metabolism disorders and can reduce CREA,UREA levels to improve renal function;2.Apoptosis and autophagy are involved in the development of vascular calcification in rats with chronic kidney disease,and excessive autophagy increases vascular calcification.Bushen Huoxue Decoction can activate the Akt signaling protein in the aorta of chronic kidney disease rats,increase the expression of Bcl-2 protein and decrease the expression of Bax and Caspase-3 apoptotic proteins.It can also activate the signal protein m-TOR and inhibit the expression of autophagy-related proteins.The calcification of aorta in rats with chronic kidney disease can be inhibited by regulating apoptosis and autophagy.3.High phosphorus can successfully induce the calcification of vascular smooth muscle cells in a dose-dependent manner.The apoptosis inhibitor can inhibit the apoptosis and calcification of smooth muscle cells in a concentration-dependent manner,indicating that apoptosis is involved in the calcification of vascular smooth muscle cells,and ROS May mediate the apoptosis.of vascular smooth muscle cell at its early stage.4.Bushen Huoxue Decoction serum can reduce calcium deposition form calcification of vascular smooth muscle cells in a concentration-dependent;its mechanism includes:?To Inhibit the vascular smooth muscle calcification,the inhibition of the expression of apoptotic proteins should be promoted by activating the activity of Akt signaling protein,?To reduce calcification on vascular smooth muscle cells,the expression of autophagy should be inhibited by activating the activity of mTOR protein.At the same time Bushen Huoxue Decoction can be antioxidant by reducing the production of ROS. |
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