FSTL1 Inhibits Bone Marrow Mesenchymal Stem Cells Osteogenesis In Inflammatory Conditions

Objective: To investigate whether follistain like protein 1(FSTL1)can affect the osteoblastic function of bone marrow mesenchymal stem cells in an inflammatory environment.Methods:(1)Human bone marrow stromal cells(h BMSCs)were cultured in vitro.Through CCK-8 experiments,it was tested whether recombinant human FSTL1(rh FSTL1)protein cultured in vitro would affect h BMSCs’ proliferation activity.Rh FSTL1(rh FSTL1)protein was administered in an inflammatory environment.After osteogenesis induction,staining with alkaline phosphatase(ALP)and alizarin red was performed to detect the mineralized osteogenesis of BMSCs.The expression of osteogenesis-related genes was detected by real-time quantitative PCR.(2)BMSCs were transfected with lentivirus to inhibit or enhance the expression of FSTL1 in cells,and the transfection efficiency of lentivirus was detected by real-time quantitative PCR.In the inflammatory environment,osteogenesis was induced in BMSCs after transfection.The mineralization and osteogenesis of BMSCs were detected by ALP and alizarin red staining.The expression of osteogenesis-related genes was detected by realtime quantitative PCR.(3)The femoral heads of FSTL1 knockout mice and wild-type(WT)mice were photographed with Micro-CT,and the bone structure parameters were analyzed by software.Results:(1)The results of CCK-8 showed that treating with rh FSTL1 protein at a concentration of 0,50,and 100 ng / m L in the complete medium did not affect the proliferation activity of h BMSCs.ALP,alizarin red staining and real-time quantitative PCR showed that rh FSTL1 in vitro inhibited the ALP activity and mineralization of h BMSCs in an inflammatory environment,and reduced the expression of type-1collagen(COL1),osteopontin(OPN)and osteocalcin(OCN).(2)The results of realtime quantitative PCR showed that the over-expressing FSTL1 lentivirus(has-LVFSTL1)and the knock-down FSTL1 lentivirus(has-FSTL1-RNAi)can be enhanced or inhibit FSTL1 expression in mouse BMSCs compared with the negative control lentivirus(has-NC).ALP,alizarin red staining,and real-time quantitative PCR showed that in an inflammatory environment,the ALP activity and mineralization ability of mouse BMSCs that knocked down FSTL1 and the RNA expression levels of COL1,OCN,and OPN were higher than those of the overexpression group.(3)Compared with WT mice,FSTL1 gene knockout mice have a bone volume / total volume(BV / TV),trabecular thickness(Tb.Th),and trabecular bone pattern factor(Tb.Pf)value is higher(P <0.05),the trabecular separation(Tb.Sp)value is lower(P <0.05).The results show that FSTL1 knockout mice have more femoral skulls,higher trabecular thickness,smaller trabecular space and less osteoporosis than WT mice.Conclusion: In the inflammatory environment containing 10 ng / ml TNF-α,the increase of FSTL1 content inhibits the osteogenic ability of osteoblasts.Inhibition of FSTL1 can effectively reduce the effect of inflammation on osteoblasts.Micro-CT analysis showed that FSTL1 condition knockout mice had more femoral skulls and less osteoporosis than wild-type mice.Considering that FSTL1 is a pro-inflammatory protein,conditional knockout of this gene reduces the effect of inflammation on bone regeneration in mice.Our research shows that FSTL1 is an osteogenic inhibitor of BMSCs in an inflammatory environment and may become a new target for bone regeneration therapy in the future.