Effects Of Tougu Xiaotong Capsule On Proliferation And Differentiation Of TNF-α Injuried Osteoblasts

ObjectiveTo observe the effects of Tougu Xiaotong Capsule (TXC) on the proliferation and differentiation of osteoblasts (OBs) injured by tumor necrosis factor alpha (TNF-α), exploring the mechanism of TXC on bone formation, thus to illustrate the effects of TXC on subchondral bone remodeling.Methods1. Effects of TXC on the proliferation of TNF-αinjuried OBs1.1MTT was used to determine the concentration of TXC.1.2Groups:cells was classified into three groups, including normal (N-group), TNF-α,(TNF-α-group), and TNF-α+TXC (TXC-group).1.3Detection indexes:(1) MTT colorimetric assay was used to detected the cell viability.(2) Observation of morphology:②Inverted microscope was used to observed cell structure;②Laser scanning confocal microscope (LSCM) was used to observed the cell mortality (Hoechst33258stained the live cells, and PI stained the dead cells).③Transmission electron microscope (TEM) was used to observed the cell ultrastructure.2. Effects of TXC on the differentiation of TNF-a injuried OBs2.1Groups:cells was classified into N-group, TNF-α-group and TXC-group.2.2Detection indexes:(1) After treating with TNF-α and TXC for14days, the supernatant was collected every2days at-20℃. Alkaline phosphatase activity (AKP) activity was analyzed using colorimetrically method according to the manufacture’s instructions. The secretion of osteocalcin (OCN) was performed by using ELISA kit according to the manufacture’s instructions.(2) Mineralized nodules staining:alizarin red and tetracycline staining were used to observe the mineralized nodules of groups.(3) Scanning electron microscope (SEM) was used to observe mineralized nodules, and the calcium content was analysised by EDS at the same area and low magnification. Simultaneously, the calcium content of signal cell and its surrounding was detected.Results1. Effects of TXC on OBs proliferation 1.1Cell proliferation rate arrived maximum after treated with1.25mg/mL TXC for24hours1.2Cell viability of TXC-groups were higher than TNF-α-group, and1.25mg/mL TXC-group was best.1.3OBs of TNF-α group became smaller, contractive, and the majority of cells became round, dieing. However, the cell morphology of TXC-groups were improved, and the1.25mg/mL TXC-group was best.1.4Cell mortality of TNF-a group arrived to55.07%. However, the cell mortality of TXC-group reduced to18.68%.1.5Many cell ultrastructure, including heterochromatin and membrane in nuclear, and endoplasmic reticulum, was abnormal in TNF-a group. Besides, some cells appeared apoptosis. While ultrastructure of TXC-group was improved.2. Effects of TXC on cell differentiation2.1Compared with TNF-α group, TXC promoted the expression of AKP and OCN after2days, and the promotion was obvious after6days.2.2Mineralized nodules were negative by alizarin red and tetracycline staining of TNF-a group. However, mineralized nodules were found in N-group and TXC-group.2.3Mineralized nodules of N-group and TXC-group were observed by SEM, but TNF-α group was negative. Calcium content of TXC-group were higher than TNF-α group at the same area and low magnification. What’s more, the calcium content and Ca/P atomic number of signal cell and its surrounding were higher.Conclusion1. TXC promoted Obs proliferation and protected Obs from TNF-α inhibition.2. TXC promoted Obs differentiation, maturation and mineralization, and and protected Obs from TNF-α inhibition.3. TXC protects Obs against TNF-α injuries.4. TXC protects bone formation against TNF-α injuries, that is a mechanism of TXC on subchondral bone remodeling.