Experimental And Clinical Study On Protein Expression Of The Human Osteoblast-like Cell Which Intervened By Alcohol And Treated By The Invigorating The Kidney Method

1 Objective of researchOsteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of active constitute. It was a familiar type that caused by alcohol. The pathogenesis and control technique of this type needed continual study. Researches on its pathogenesis and prevention and cure by Chinese medicine were important.The over intake of alcohol was the first cause of Osteonecrosis of the femoral head caused by alcohol. Its pathogenesis included cell toxic action and fatty degeneration and necrosis of osteocyte.The basic pathological change of osteonecrosis was the losing of banks of osteoblast on the surface of bone trabecula and the blankness of lacuna in osteocyte. There was no ideal method for the prevention and cure of osteonecrosis of the femoral head by the method of western medicine.The kidney being in charge of the bone was the basic conception in the treatment on osteopathy by traditional Chinese medicine. There had an extensive use for the treatment on alcoholic osteonecrosis of the femoral head by the method of invigorating the kidney. But its therapeutic mechanism was not well formulized with modern scientific means.Proteomics was a frontier in studying protein groups. It was mainly investigate the characters of proteins, thus we could know the cellular functions, physiological and biochemical process and pathogenic process at the level of proteins. The pathogenesis, pharmaceutical target point and new drugs could be found by comparing the express of proteins both in normal cells and sicken cells, and in sicken cells and medicated sicken cells. Proteomics provided a conceptual technical platform for investigating the syndrome differentiation in TCM.From the point of view of modern technology, there was a genic or protein essence in the treatment of this disease by invigorating the kidney and invigorating the marrow. This study processed with cellar experiment in vitro. We simulated the model of alcoholic osteonecrosis of the femoral head. Afterwards, cells were treated with pastille serum of bolus of six drugs including rehmannia that was a reprehensive prescription of the methods of invigorating the kidney. Then we used two-dimensional electrophoresis techniques to find out the differential protein points after the intervention of alcohol and treatment of pastille serum. The preliminary analysis established basement for discovering the structural function of the differential protein and pathogenesis of this disease and therapeutic mechanism of the treatment of invigorating the kidney.2 Methods and results2.1 The effect of the method of invigorating the kidney on cellar proliferation of human osteoblast-like cell 0S-7322.1.1 Drafting of culler curve of growthWe selected human osteoblast-like cell 0S-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 7 days continuously. The control group was with the blank serum. Then the cell counting and curve of growth drafting was done. Results showed that there was better effect of cell proliferation in the treatment groups than in the control group. There was strong effect in both the high dosage treatment group and the medium dosage treatment group, among them the medium dosage treatment group showed the stronger effect. The logarithmic phase was 3 to 6 days.2.1.2 The proliferation experiment on the human osteoblast-like cell OS-732 with the method of MTT We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 5 days continuously. The control group was with the blank serum. The optical concentration (OD) was tested daily. Results showed that there was no significant difference in the 24 hours groups and 48 hours groups. But in the 3 days groups, 4 days groups and 5 days groups, there was stronger effect of cell proliferation in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect of cell proliferation.2.1.3 Experimental study on the secretion of alkaline phosphatase (ALP) of human osteoblast-like cell OS-732We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 3 days, 4 days and 5 days respectively. The control group was with the blank serum. Then alkaline phosphatase kit and Elisa Machine was used to test ALP and optical concentration.Results showed that there was stronger effect of cell proliferation higher ALP in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect.2.2 Experimental study of alcohol intervention and pastille serum treatment on human osteoblast-like cell OS-7322.2.1 Detection of apoptosis rate of human osteoblast-like cell OS-732 intervened by alcohol with different concentrationsWe selected the human osteoblast-like cell OS-732 in good condition and added different concentration of alcohol. The same amount of RPMII640 medium was added into the control group. Cells were collected after 24 hours, 48 hours and 72 hours intervention. Then we used flow hyetometer (FCM) to test after cool alcohol fixed and PI liquid stained. Results showed that there was higher apoptosis rate in the intervention group than that in the control group. It had significant meaning in statistics (p＜0.05). The apoptosis rate had positive correlation with the alcohol concentration and the acting time of alcohol. 2.2.2 Electron microscopic observation of the human osteoblast-like cell OS-732 intervened by alcoholIt was divided into the normal group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Results of electron microscopic observation showed that the normal human osteoblast-like cell OS-732 was with relatively great and polymorphous nucleolus and was with apparent nucleoli. There was quite a lot of ribosome, golgis apparatus and lysosome-like grain. Changes took place in some cell organ after the intervention of alcohol, which was the reducing of ribosome and endoplasmic reticulum and more bubble, The typical apoptosis such as karyopyknosis could be seen. There was rehabilitation, for example, the endoplasmic reticulum and golgis apparatus was visible, in the cell organ after the treatment of pastille serum.2.3 Preliminary analysis of differential protein after the intervention of human osteoblast-like cell OS-732 by alcohol and the treatment of invigorating the kidney methodThe human osteoblast-like cell OS-732 was divided into the blank control group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Total protein was extracted and separated with the two dimensional gel electrophoresis techniques. Then the electropherogram was analyzed by image analysis software. Results showed that there was five apparently different points of protein that was SSP1002, SSP 1401, SSP 1602, SSP 8501 and SSP 8504 after comparison between the intervention of alcohol group, the treatment group and the blank control group. It was low expression of protein in the intervention of alcohol group and was high expression of protein in the treatment group. The bolus of six drugs including rehmannia pastille serum could improve the expression of protein.2.4 Clinical study on the treatment of alcoholic steonecrosis of the femoral head by the invigorating the kidney methodWe compared the curative effect of the shengmai ossify group and the combination of the bolus of six drugs including rehmannia and the shengmai ossify group.69 cases of patients with alcoholic steonecrosis of the femoral head were systematically observed. Results showed that the effective rate in the combination of the bolus of six drugs including rehmannia and the shengmai ossify group was 83.78%, and that in the shengmai ossify group was 81.25 %. There was no significantly difference between two groups. But as to the pain relief, the effect of the bolus of six drugs including rehmannia and the shengmai ossify group was better than that in the shengmai ossify group (P=0.0028). The improvement of physical signs was better in the bolus of six drugs including rehmannia and the shengmai ossify group than that in the shengmai ossify group (p＜0.05).There was no significant difference in absorption of sequestra, formation of fresh bone and periosteal reaction between two groups.3 conclusionsThe pastille serum of bolus of six drugs including rehmannia had proliferation promotion, and the promotion of ALP secretion of the human osteoblast-like cell OS-732.Different concentration of alcohol could lead to apoptosis of the human osteoblast-like cell OS-732. There was positive correlation between the apoptosis and alcohol concentration. The inverted microscope and the transmission electron microscope observation showed that cells were damaged, such as cell death, apoptosis and bubble-like change in survival cells.The two-dimensional electrophoresis technique of proteomics showed that there was descending of partial protein expression in the human osteoblast-like cell OS-732 intervened by alcohol. But there was ascending of partial protein expression in the human osteoblast-like cell OS-732 treated by bolus of six drugs including rehmannia serum.The bolus of six drugs including rehmannia was the reprehensive prescription of the method of invigorating the kidney. This prescription had better curative effect on the alcoholic steonecrosis of the femoral head in clinic. The combination of activating blood circulation to dissipate blood stasis could improve curative effect in clinic.