The Effect Of Diterbutyl Phthalate Extracted From Panax Notoginseng On Osteoarthritis And Its Mechanism

BackgroundOsteoarthritis(OA)belongs to the most prevalent form of arthritis characterized by a chronic and irreversible course.Currently,the pathogenesis underlying OA remains unclear,there are also no satisfactory managements to abolish OA progression.Recently,the essential role of subchondral bone in OA has attracted increasing attention.Subchondral bone and articular cartilage constitute a structural and functional unit.Subchondral bone not only offers a mechanical support for articular cartilage,but also responds quickly to the mechanical force applied on the joint surface.During OA initiation,mechanical instability of the joint triggers the abnormal activation of osteoclastogenesis in subchondral bone,leading to accelerated bone turnover rate and elevated bone loss at the early stage of OA,which further exacerbates OA progression.Moreover,osteoclast-mediated neovascularization in subchondral bone results in aberrant angiogenesis and joint pain.Therefore,inhibiting the overactivation of subchondral osteoclastogenesis could be a potential approach in the treatment of OA.Panax notoginseng(PN)refers to a natural plant with multiple biological properties including anti-inflammation,promoting bone healing,hemostasis,acesodyne,neuroprotection and anti-osteoporosis.However,the components of PN are complex and changeable.Whether there is an active ingredient of PN to regulate osteoclastogenesis with a therapeutic effect on OA,and the mechanisms by which it takes effect remains to be elucidated.In this study,we found that Diterbutyl Phthalate(DP),an active component with a high affinity with BMMC membrane,was identified from PN through two-dimensional/bone marrow mononuclear cells/cell membrane chromatography/C18column/time-of flight mass spectrometry(2D/BMMC/CMC/C18/TOFMS).In vitro we investigate the influence of DP on osteoclastogenesis.In vivo we determine the therapeutic role of DP in ACLT-induced OA mice model.Moreover,we explore its related molecular mechanisms.ObjectivesPart 1:To identify the active ingredient of PN with a high affinity with BMMC membrane,and verify its effect on osteoclastogenesis in vitro.Part 2:To elucidate the effect of DP extracted from PN on the abrasion of articular cartilage and the microstructural alterations of subchondral bone in ACLT-induced OA mice model.Part 3:To investigate the effect of DP screened from PN on the abnormal subchondral bone angiogenesis and joint pain in ACLT-induced OA mice model.Part 4:To demonstrate the potential mechanisms by which DP plays a role in treating OA.MethodsPart 1:(1)The concentrated solution was extracted from PN and the primary BMMC were harvested from the mice femora.(2)2D/BMMC/CMC/C18/TOFMS system was carried out to identify the active component(DP)from PN with a high affinity with BMMC membrane.(3)Cell Counting Kit-8(CCK-8)was employed to confirm the viability of BMMC and RAW264.7 cells with different concentrations of DP intervention.(4)Osteoclasts were induced from BMMC and RAW264.7 cells with RANKL,which were underwent DP intervention.(5)The effect of DP on the RANKL-induced osteoclastogenesis from BMMC and RAW267.4 cells was detected by TRAP staining.Part 2:(1)OA mice models were achieved with anterior cruciate ligament transection(ACLT)to mimic the mechanical instability,which were underwent DP intervention.(2)Hematoxylin and Eosin(HE)staining were performed to investigate the degeneration of articular cartilage.(3)Safranin O and fast green(Safranin O)staining was used to show the loss of proteoglycans in articular cartilage.(4)Osteoarthritis Research Society International(OARSI)score was carried out to quantitatively analyze the abrasion of articular cartilage.(5)Immunohistochemistry(IHC)and Immunofluorescence(IF)staining were employed to detect the expression of articular cartilage metabolic markers,including Matrix Metalloproteinase 13(MMP-13),Collagens X(Col-X)and Lubricin.(6)Micro-CT was adopted to observe the alterations of subchondral bone microstructure and relative bone parameters,including Bone volume/tissue volume(BV/TV),Trabecular separation(Tb.Sp),and Subchondral bone plate thickness(SBP.Th).(7)TRAP staining was utilized to observe the osteoclastogenesis in subchondral bone.(8)Western Blotting(WB)was performed to examine the effect of DP on the expression of Cathepsin K(CTSK)during RANKL-induced osteoclastogenesis.Part 3:(1)OA mice models were achieved with ACLT operation to mimic the mechanical instability,which were underwent DP intervention.(2)Micro-CT angiography was carried out to observe the alterations of subchondral angiogenesis and the vessel parameters,including vessel volume(VV)and vessel number(VN).(3)IF staining was employed to detect the expression of H-type vessel(CD31^hiEMCN^hi)and Matrix Metalloproteinase 2(MMP-2)in subchondral bone,which were two indicators of angiogenesis.(4)IF staining was adopted to observe the expression of TRAP/Netrin-1colocalization and CGRP in subchondral bone,which were two markers of neurogenesis.(5)Von Frey test and Plantar test were used to examine the pain-related behavioral phenotypes.(6)WB was performed to investigate the effect of DP on the expression of Netrin-1 during RANKL-induced osteoclastogenesis.Part 4:(1)Osteoclasts were induced from RAW264.7 cells with RANKL,which were underwent DP intervention.(2)Dil staining was employed to determine the effect of DP on the RANKL-induced osteoclasts fusion.(3)IF staining was adopted to examine the effect of DP on the nuclear translocation of NFATc-1 during RANKL-induced osteoclastogenesis.(4)WB was used to explore the effect of DP on the activation of ERK/c-fos/NFATc1 signaling pathway during RANKL-induced osteoclastogenesis,and the expression of DC-STAMP and Atp6v0d2,which were two key modulators for osteoclasts fusion.ResultsPart 1:(1)DP is an active component identified from PN,demonstrating a high affinity with BMMC membrane.(2)DP showed no significant toxicological effect on BMMC and RAW264.7 cells proliferation when its concentration was blow 20??.(3)DP notably inhibited the RANKL-induced osteoclastogenesis from BMMC and RAW264.7 cells in vitro.Part 2:(1)He staining indicated that DP significantly inhibited the ACLT-induced hyaline cartilage(HC)thickness decline and calcified cartilage(CC)thickness elevation.(2)Safranin O staining showed that DP notably suppressed ACLT-induced proteoglycan loss of articular cartilage.(3)OARSI displayed that the histopathological score of knee joint was substantially reduced in DP group in relative to ACLT group.(4)IHC and IF staining illustrated that DP apparently increased the expression of Lubricin and decreased the expression of Col-X and MMP-13 when compared with ACLT group.(5)Micro-CT showed that DP significantly increased BV/TV and SBP.Th and diminished Tb.Sp in subchondral bone in relative to ACLT group.(6)TRAP staining demonstrated that DP drastically abrogated the ACLT-induced abnormal activation of osteoclastogenesis in subchondral bone.(7)WB indicated that DP significantly attenuated up-regulated expression of CTSK during RANKL-mediated osteoclastogenesis.Part 3:(1)Micro-CT angiography showed that DP notably reduced VV and VN in subchondral bone in relative to ACLT group.(2)IF staining illustrated that the expression of CD31^hiEMCN^hi and MMP-2 in subchondral bone was apparently down-regulated in DP group when compared with ACLT group.(3)Von Frey test and Plantar test showed that DP significantly alleviated the joint pain in ACLT-induced behavioral changes.(4)IF staining displayed that DP substantially abrogated the expression of TRAP/Netrin-1 colocalization and CGRP when compared with ACLT group.(5)WB indicated that DP drastically attenuated the up-regulated expression of Netrin-1 during RANKL-induced osteoclastogenesis.Part 4:(1)Dil staining showed that DP significantly inhibited the RANKL-mediated osteoclasts fusion.(2)IF staining indicated that DP notably repressed the nuclear translocation of NFATc1 during RANKL-mediated osteoclastogenesis.(3)WB demonstrated that DP apparently attenuated the activation of ERK/c-fos/NFATc1 signaling pathway and abrogated the up-regulated expression of DC-STAMP and Atp6v0d2 during RANKL-induced osteoclastogenesis.Conculsion(1)DP is an active ingredient recognized from of PN with a significantly inhibitory effect on RANKL-induced osteoclastogenesis in vitro.(2)DP restored the normal microstructure of subchondral bone and attenuated the degeneration of articular cartilage in ACLT-induced OA mice model in vivo.(3)DP retarded the abnormal angiogenesis in subchondral bone and relieved the joint pain in ACLT-induced OA mice model in vivo.(4)DP blocked osteoclastogenesis by suppressing osteoclasts fusion via inhibiting RANKL-induced ERK/c-fos/NFATc1 pathway.In summary,we demonstrate that DP might serve as a promising pharmacological candidate to prevent OA onset and progression.