Mechanism Of TSP-1in Thigh Bone’s Necrosis

Objective: Osteonecrosis (ONFH) is a common illnesses. Hip trauma is result in Traumatic ONFH,while excess hormones and alcohol often cause the non-invasive ONFH. Nowadays SANFH ranks the firstof non-invasive ONFH. SANFH (Steroid-induced avascular necrosis of femoral head, SANFH) has a slowprogress on clinical treatment, high morbidity and bad the lives quality of patients. Because itspathogenesis is unclear,the effective cure-method lacks. Currently SANFH remains a worldwide problemin the orthopedic field. Endotoxin can induce vascular inflammatory response, cause vascular endotheliuminjury,and makes blood become hypercoagulable state. At last, a large number of hormones induceosteonecrosis on those factors. This progress is similar to the pathogenesis of SANFH. TSP-1which is thefirst discovered polypeptide is endogenous angiogenesis inhibitor. TSP-1can inhibit angiogenesis byinhibiting the combination of angiogenic growth factor and its receptor in the endothelial cell surface.Currently, the important role of TSP-1in the anti-angiogenic pathological process has been confirmed bynumerous experiments. How is the role of TSP-1in femoral head necrosis? It is unclear that SANFH isknocked down. First, we use endotoxin (LPS) and hormone to induce femoral head necrosis in mice.Second, we take a comparison of femoral head necrosis of TSP-1knockout mice and wild-type mice. Atlast, the ultimate aim is to explore the mechanism of TSP-1in femoral head necrosis in mice.1. Toinvestigate the expression of TSP-1gene on necrosis of the femoral head.2. To investigate the protectiveeffect of TSP-1gene on necrosis of the femoral head.3. To explore the effect of TSP-1gene on tumornecrosis factor (TFN-а) and interleukin1(IL-1β).Methods: Firstly, we selected twenty6-week-old C57BL/6mice, and they were randomly dividedinto two groups (n=10, A, B). Group A was a control group, group B is stimulated by LPS and hormone(endotoxin (LPS)+steroid, endotoxin (LPS)+hormone) group, group C is TSP-1KO group (n=6).Group B and C mice were injected endotoxin(LPS) by intraperitoneal with50ug/kg.We injected one byone day in two days. The second injection of endotoxin (LPS) was in other day by20mg/kg dose ofmethylprednisolone,1time a day for three days.In group A was injected saline. After the injection of LPSwas executed each4mice of group A and B, we observe whether model is successfully established, Femoral head and liver HE staining, immunohistochemical staining, immunohistochemistry, IHC) wereobserved. mRNA which was contained in liver tissue was extracted, used to do PCR, and detected the TSP-1gene expression.6weeks for executing A, B, C groups all mice, femoral head and liver HE staining,immunohistochemical staining to observe, three groups of mice liver tissue extract mRNA in PCR,detecting the expression of TNF alpha and beta IL-1. Using SPSS17.0software to analyze theexperimental results. Data was compared with single factor analysis of variance between groups,(P <0.01)for significant differences, statistically significant;(P <0.05) as there are differences, statisticallysignificant;There was no significant difference (P>0.05), no statistical significance.Results: After three weeks, HE staining was used to observe A and B group mice. We found thatgroup A is normal liver, liver tissue structure is clear.Femoral trabecular bone integrity, rules, bone cells canbe clearly seen, marrow fat cells is relatively small, between normal form. Group B liver cell swelling,visible small fat vacuoles in cells, liver cable structure is not clear;Femoral trabecular bone is normal andpart of the fracture, some bone cell edge, pyknosis, marrow fat cells between diameter increases, thenumber increased;To the immunohistochemical observation, we found more granules in group A(osteoprotegerin, OPG), less bone protection element ligand (osteoprotegerin ligand, OPGL)brownish-yellow granules, not more positive staining rate.Two groups of liver mention mRNA TSP PCR todetect group B-1gene expression was significantly higher in group A (P values <0.05).6weeks forexecuting A, B, C groups all mice, HE staining observation:A set of normal liver, liver tissue structure isclear;Femoral trabecular bone integrity, rules, bone cells can be clearly seen, marrow fat cells is relativelysmall, between normal form. Group B liver cells visible uneven fat vacuoles, fuzzy liver tissuestructure.Trabecular bone thinning, femoral head fracture, bone necrosis empty pit increased, bone marrowbetween fat cells. Group C liver and femoral head does not change significantly than group A.Empty bonepit B group was obviously more than A and C group (P <0.01), A, C, no significant difference (P>0.05).Immunohistochemical observation: A group of3and6weeks of bone protection (osteoprotegerin, OPG)see more tan granules, bone protection element ligand (osteoprotegerin ligand, OPGL) brownish-yellowgranules is less, positive staining rate has no obvious change; Group B OPG positive rate of6weeks is3weeks, OPGL higher positive rate of6weeks is3weeks.Group C OPG tan see more particles, OPGL tanless particles.Group C OPG positive expression is slightly lower in group A, OPGL positive expression is slightly higher in group A. Group B compared with group A and C, femoral head OPG local positiveexpression decreased obviously, local positive rate increased expression of OPGL, have obvious difference(P <0.01).Local positive rate between group A and C OPG expression was slightly lower, OPGL localpositive expression slightly increased, no significant difference (P>0.05). Three groups of liver extractmRNA for PCR detection of TNF-а, IL-1gene expression.B and C group were higher than group A (P <0.05), group C than group B (P <0.05).Conclusion:1The TSP-1gene expression of SANFH is higher after necrosis of the femoral head than normal mice.This implies that TSP-1gene expression has a positive correlation with vascular necrosis of femoral head.2.TSP-1knockout of steroid-induced osteonecrosis has a protective effect that it can protect bone cellsand prevent the occurrence of femoral head necrosis.3. TSP-1knockout inhibits the expression of pro-inflammatory factors (TNF-а, IL-1β).Conclusion: TSP-1gene knockout can improve the blood supply to the femoral head, reduceischemic symptoms, promote bone metabolism, protect bone cells to prevent the occurrence of femoralhead necrosis and reduce pro-inflammatory cytokines (TNF-а, IL-1β) inflammation. We expect thatexpression of TSP-1gene can inhibit bone cell apoptosis by inhibiting the femoral head necrosis. At last,TSP-1gene may plays a role as early prevention of steroid-induced osteonecrosis.