The Role Of Ursolic Acid As A Inhibitor Of PTP-oc On Osteoclastogenesis And Root Resorption During Orthodontic Tooth Movement

Orthodontic tooth movement is induced by mechanical stimuli and facilitatedby remodeling of the periodontal ligament(PDL) and alveolar bone. However, Whenthe orthodontic force is put on the dislocated teeth, it is inevitable the occurrence ofdifferent extents of root resorption that is one of the most common complicationsduring orthodontic treatment. Although mild root resorption caused by orthodontictreatment would repair by itself, severe root resorption can affect the stability and thehealth of teeth and have a negative impact on orthodontic treatment. Root resorptionand bone resorption mechanisms are similar, the odontoclasts shares similarcharacteristics with osteoclasts. At present, there are not effective therapeuticalmethods at present, therefore, how to avoid and reduce root absorption have becomethe problem which is looking forward to be solved by the orthodontic doctors.Protein Tyrosine Phosphatases(PTPs) are key enzymes in the process of cellsignaling, which are closely related to physiological and pathological processes ofhuman. Protein tyrosine phosphorylation of proteins is bound up with osteoclastproduction, adhesion and activity, as illustrated by the pivotal role of the Src PTK inthese cells. Phosphorylation also plays key roles in signaling pathways of osteoclast.Osteoclastic protein tyrosine phosphatase(PTP-oc) belongs to the receptor PTPfamily and is specifically expressed in precursors of osteoclasts, including Blymphocytes, cells of the monocyte-macrophage lineage and mature osteoclasts; it isan activator of osteoclast differentiation and activity. PTP-oc may be a potentialtarget for reducing the root resorption by inhibiting its activity. To test the hypothesis,a large number of researches were carried out.1. Expression of human osteoclastic protein-tyrosine phosphatase catalyticdomain in Escherichia coliHuman osteoclastic protein-tyrosine phosphatase(PTP-oc) gene serves as a template in our study. Then, human osteoclastic protein-tyrosine phosphatasecatalytic domain(ΔPTP-oc) gene has been amplified by PCR; pMD18-ΔPTP-oc andpET-28a-ΔPTP-oc have been constructed, which were consequently cut by Nco I andXho I and identified by agarose gel electrophresis. Finally, pET-28a-ΔPTP-oc hasbeen transformed into E.coli BL21(DE3) so as to get cell lines that express targetprotein at a high level.2. Purification and characterization of human osteoclastic protein-tyrosinephosphatase catalytic domain in Escherichia coliΔPTP-oc was expressed in E.coli BL21(DE3) cells as a fusion with asix-histidine tag and was purified via Ni-NTA agarose affinity chromatography. Thepurity of the sample is more than80%by the identification with SDS-PAGE. Theresult of CD spectra is in a good agreement with the secondary structure prediction bythe use of SOPM, which proved the recombinant ΔPTP-oc have the correct spatialstructure. The kinetics of enzymatic reactions showed that, when p-NPP serves as thesubstrate, its Km=801.8μmol, Vmax=6.1μmol/min. The study of characteristics ofthe enzyme has demonstrated that the optimal reaction temperature ΔPTP-oc is34℃,the optimal ionic strength is0, which are similar to other PTPs. But the optimal pHvalues(pH7.0) of ΔPTP-oc is different from other PTPs.3. The screening of ΔPTP-oc inhibitorsWe established the detection methods for the enzyme. It is chromogenicsubstrate method. There are four kinds of monomer compound detected from76monomer compounds. They had obvious inhibitory to ΔPTP-oc, in which theinhibitory effect of Ursolic acid reached98.3%, with the half maximal inhibitoryconcentration (IC50) was6.381±0.56μm/L. We found that Ursolic acid was acompetitive inhibitor of ΔPTP-oc with the inhibition constant(Ki) was7.9μm/L. Inaddition, Ursolic acid showed a more efficient inhibitory potency against ΔPTP-octhan other PTPs in vitro. Therefore, we selected Ursolic acid as a further researchobject, and researched the effects of Ursolic acid on osteoclastogenesis and activity.4. The role of Ursolic acid in differentiation and activity of human monocytic U937cell-derived, osteoclast-like cellsBy1×10-7M/L TPA and1×10-8M/L1,25(OH)2D3treatment, U937cells weredifferentiated into osteoclast-like cells. CCK-8results showed that Ursolic acid doesnot affect osteoclast differentiation of U937cells in less than5μmol. Therefore, U937cells were treated with different concentration of Ursolic acid (1μm,2.5μm and5μm).A obvious dose-dependent increase in the c-Src PY-527levels and the expression ofc-Src constant were found by Western blot methods. TRAP staining showed that,compared with the control group, Ursolic acid can significantly inhibit thedifferentiation of U937cells to osteoclast in a dose-dependent manner. Quantitativegene expression analysis for osteoclastic marked genes(TRAP, RANK, MMP-9, CKand CTR) was performed by Real time quantitative PCR. Compared with the controlgroup, the marked genes of treated with different concentrations of Ursolic acidgroups were significantly inhibited in a dose-dependent manner.5. Effects of Ursolic acid on root resorption during experimental toothmovementThe8weeks male Wistar rats were randomly divided into control group, normalgroup, model group, and model group were divded into low-dose group(0.5mmol),medium-dose group(1mmol), high-dose group(2mmol).50g mesial force wereapplied to the maxillary first molar by nickeltitanium closed-coil springs for14d and28d. Tooth movement distance of the maxillary first molar was measured, and thetooth movement of28d high-dose group were less than that of the control group. Theperiodontal tissue morphology results show that, as the concentration of Ursolic acidincreased, root resorption appears delayed and the extent of root resorptionweakened. Immunohistochemical staining results showed that, compared with thecontrol group, c-Src Tyr527phosphorylation of model groups increased with theconcentration of Ursolic acid increased. The Ursolic acid treatment inhibited the rootresorption and increased c-Src Tyr527phosphorylation in a dose-dependent manner.According to the results above, we can draw the following conclusions:1. The soluble recombinant expression of ΔPTP-oc is feasible. Whereby a decrease in the induction temperature gave rise to a higher solubility ofrecombinant protein.2. ΔPTP-oc was expressed in E. coli cells as a fusion with a six-histidine tagand was purified via Ni-NTA agarose affinity chromatography. Comparedwith other PTPs, ΔPTP-oc is different, such as the optimal pH is7.0, theoptimal reaction temperature ΔPTP-oc is34℃.3. Ursolic acid showed an efficiently competitive ΔPTP-oc inhibitory potencyin vitro.4. By inhibit of ΔPTP-oc, Ursolic acid could increase c-Src PY527levels,inhibit the c-Src signaling. Ursolic acid may play an important inhibitor rolein differentiation and activity of human monocytic U937cell-derived,osteoclast-like cells.5. Ursolic acid could reduce the resorption craters areas in a dose-dependentmanner.