The Biological Effect Study Of Salvia Miltiorrhiza Injection On The Human Chondrocytes And Osteoblasts In Vitro

Objective:This study was to in vitro investigate the biological effects of salvia miltiorrhiza injection(SMI)on the primary human articular chondrocytes and osteoblasts.The mechanism of the treatment of osteoarthritis and osteoporosis was discussed.Methods:The concentration of SMI used in experiment was calculated by Salvia miltiorrhiza Bge(1.5 mg·mL-1).The control group of chondrocytes was cultured with dulbecco’s modified eagle medium/F12 medium containing 10%Fetal bovine serum,while the experimental groups were administrated with 0.5,1.0,2.0,4.0 mg-mL-1 of SMI.The experimental groups of osteoblasts were administrated with 0.5,1.0,2.0,3.0 mg·mL-1 of SMI,and the control cells without SMI administration.The indicator cell assay(DNA staining method)was used to detect the mycoplasma contamination of chondrocytes and osteoblasts;The RT-PCR assay was used to detect the expression of COL1A1,COL2A1 mRNA.ALP activity detection and Alizarin red stain for calcium of osteoblasts were used to identify osteoblasts;The Methylthiazole tetrazolium(MTT)assay was used to evaluate cell proliferation activity of the third passage human articular chondrocytes and human osteoblasts;The mRNA expression level which was to evaluate cell differentiation of collagen type Ⅰ/Ⅱ of human articular chondrocytes and BMP-2,Runx2,OCN,COL1A1,BSP of human osteoblasts were determined by real time reverse transcription-polymerase chain reaction(RT-PCR);Enzyme-Linked immunosorbent assay(ELISA)was used to assess the content of collagen typeⅠ,Ⅱ of human articular chondrocytes and collagen typeⅠ,OPN,OCN of human osteoblasts;The total content of glycosaminoglycan(GAG)in cells were determined using glycosaminoglycan(DMMB)kit;Alkaline phosphatase activity in osteoblasts was detected by alkaline phosphatase(ALP)kit.Results:1.Mycoplasma test and Cell identification:The results showed that the human primary articular chondrocytes and osteoblasts were not found the mycoplasma contamination,under the results of mycoplasma positive control and negative control were acceptable;The results of COL1A1,COL2A1 mRNA to be detected indicated that the cells were cartilage specific cells;The results of ALP activity and calcium nodules to be assayed indicated that the hFOB1.19 had osteogenic capacity.2.The biological effects of primary articular chondrocytes:The MTT assay showed that the cell proliferation in all of SMI administration groups were lower than that in the control group,and with dose-dependent manner,indicating that SMI had inhibition effect on proliferation of the primary human articular chondrocytes;The mRNA expression level of collagen type I in all of SMI administration groups were lower than that in the control group with a dose-dependent manner;The collagen type I protein content in the groups with lower concentration(≤2.0 mg·mL-1)of SMI was significantly decreased,compared with that in the control group.The results suggested that the lower concentration of SMI may have a role in preventing de-differentiation of chondrocytes.Meanwhile,lower concentration(<4.0 mg·mL-1)of SMI increased the content in GAG of the primary human chondrocytes,indicating that SMI has effect on chondroregeneration.3.The biological effects of osteoblast:The MTT assay showed that the osteoblast proliferation in SMI(4.0,6.0,8.0,10.0 mg·mL-1)administration groups were lower than that in the control group,and with dose-dependent manner.The groups with lower concentration(<4.0 mg·mL-1)of SMI had no effect on cell proliferation,compared with that in the control group;Fluorescence quantitative RT-PCR showed that 2.0 mg·mL-1 of SMI administration group increased the expression of BMP-2,Runx2,OCN,COL1A1,BSP mRNA at 3 days culture.But at 7 days culture,these gene expressions had no significant difference compared with that in the control group;The result of ALP showed that the activity of ALP in all of SMI administration groups was promoted at 3 days culture with SMI,but at 7 days culture,the activity of ALP had no significant difference compared with that in the control group;The result of ELISA showed that the collagen type I protein content in the group with SMI administration at 2.0 mg·mL-1 of concentration,and the OCN content in the groups with SMI administration at 2.0,3.0 mg·mL-1 of concentration were promoted,while the OPN content in the groups with SMI administration at 0.5,1.0,2.0 mg·mL-1 of concentration had no significant difference compared with that in the control group at 3 days culture.Conclusion:1.The primary human articular chondrocytes and osteoblasts hFOB1.19 were not contaminated by mycoplasma.The cells used in this study were identified as chondrocytes and osteoblasts.2.Salvia miltiorrhiza injection could slightly inhibit the proliferation of third passage primary human articular chondrocytes;and inhibited the production of collagen type I(a de-differentiation marker)and promoted the synthesis of GAG.These results indicated that SMI may have promotive effect on chondroregeneration.3.Under no affection on the proliferation of osteoblasts,a certain concentration of salvia miltiorrhiza injection promoted the expression of BMP-2,Runx2,OCN,COL1A1,BSP mRNA,and ALP activity,and the protein content of collagen type I,OCN.These results suggested that SMI may have promotive effect on osteoblast differentiation and osteogenesis.