Helenalin Inhibits Osteoclastogenesis And Inflammatory Bone Destruction Through The NF-?B Non-canonical Signaling Pathway

Bone metabolism is a continuous,dynamic remodelling process,the homeostasis of which depends on a dynamic balance between osteoclasts(bone resorption)and osteoblasts(bone formation).Osteoclasts(OCs)are the only 'osteoclastic' cells in the body,and an increase in their number or over-activation will upset the balance between bone resorption and bone formation,leading to a variety of osteolytic diseases such as osteoporosis,rheumatoid arthritis,osteoarthritis,aseptic loosening of implants,osteomyelitis,periodontitis,malignant tumour bone metastases.Osteomyelitis,periodontitis,bone metastases from malignant tumours and multiple myeloma.Targeting osteoclasts and inhibiting osteoclast overproduction and activation is an essential strategy for treating a range of osteolytic diseases.In recent years,pharmacodynamic and pharmacological studies of natural small molecule compounds based on signalling pathways have become a hot research topic in various disease areas.Approximately 64.4% of the drugs approved by the US FDA from 1981-2019 were natural small molecule compounds and their derivatives.The NF-?B signalling pathway plays an integral role in all stages of osteoblast differentiation and activation.Because of this,in collaboration with Professor Jiake Xu's laboratory at the University of Western Australia,our laboratory has been working for many years to screen for natural small molecule compounds that target the osteoclast NF-?B signalling pathway inhibitors.We have identified a number based on the RANKL-induced NF-?B luciferase reporter gene screening system in RAW264.7 cells,combined with computer-assisted molecular docking techniques and validated by experimental data of terpene natural small molecule compounds,including sesquiterpene lactones,as potential inhibitors of NF-?B.Sesquiterpene lactones are widely found in the families Euphorbiaceae,Jukeboxaceae,Umbelliferae,Magnoliidae,Fabaceae and Asteraceae,and have antitumour,antimalarial,anti-inflammatory and antibacterial pharmacological effects.We analysed the inhibitory effects of various sesquiterpene lactone natural small molecules on RANKL-induced NF-?B transcriptional activity and found heart chrysanthemum lactone(Helenalin)had the most significant inhibitory effect.Computer-aided molecular docking results showed that Helenalin inhibited NIK activity by forming hydrogen bonds with two amino acids,Asp536 and Glu442,respectively,of NF-?B inducing kinase(NIK).Could Helenalin then act as a NIK inhibitor to block the NF-?B signalling pathway,thereby inhibiting RANKL-induced osteoclastogenesis and activation and improving bone destruction in osteolytic diseases? To answer these questions,this thesis analyses the effects of Helenalin on osteoclast differentiation and inflammatory bone destruction at the in vivo and ex vivo levels and explores the specific mechanisms of action.Part ?: Inhibitory effect of Helenalin on osteoclast production and activationWe first examined the effect of Helenalin on the activity of mouse bone marrow-derived mononuclear macrophages(BMMs)by MTS assay and found that the IC50 of Helenalin inhibited BMMs was 5.499.The effect of Helenalin on osteoblast differentiation was examined by TRAP staining.The results showed that Helenalin inhibited osteoblast differentiation in a concentration-dependent manner and that the inhibition was more pronounced when the drug was administered at an early stage of differentiation.The effects of Helenalin on the F-actin ring and bone trap formation were analysed by immunofluorescence and scanning electron microscopy,respectively,and the results showed that this small natural molecule inhibited the number and circumference of the F-actin ring in a gradient-dependent manner and significantly inhibited bone erosion.The results suggest that Helenalin significantly inhibits the bone resorption activity of osteoblasts.The effect of Helenalin on the apoptosis of BMMs was examined by flow cytometry,and the results showed that Helenalin did not affect the apoptosis of osteoclasts in the range of 0.125?M,suggesting that the effect of this small molecule compound on osteoclast differentiation and activation is not implemented through a pro-apoptotic effect.Part ?: Molecular mechanism of Helenalin inhibition of RANKL-induced osteoclast differentiation and activationBased on the results of the previous NF-?B luciferase reporter gene screening,computer-assisted molecular target docking screening,and the critical role of NF-?B signalling pathway in osteoclastogenesis and function,this thesis focuses on the molecular mechanisms of the NF-?B signalling pathway,including the classical IKK?/?/I?B?/p65 signalling pathway and the non-classical NIK /IKK?/p100/p52 signalling pathway.We first analyzed the effect of Helenalin on the classical NF-?B pathway by Western blot and immunofluorescence.We found that Helenalin had no significant effect on the degradation of I?B?,the expression of p65 and nucleation,suggesting that this natural small molecule compound does not affect the classical NF-?B signalling pathway.We then examined the effects of Helenalin on the non-classical signalling pathway.We found that Helenalin effectively inhibited NIK activity and protein levels,reduced the phosphorylation level of IKK?,inhibited the degradation of p100 to p52 and effectively inhibited Rel B entry into the nucleus,suggesting that: Helenalin significantly inhibited the RANKL-induced non-classical NF-?B signalling pathway activation of the classical NF-?B signalling pathway.The transcription factor NFATc1 is located downstream of the NF-?B signalling pathway and is required for osteoblast differentiation.Western blot and NFATc1 luciferase reporter gene results showed that Helenalin significantly inhibited the expression and transcriptional activity of NFATc1 in osteoblasts;Real-time PCR results showed that Helenalin decreased the m RNA transcript levels of osteoblast marker genes(CTSK,CTR,TRAP,NFATc1 and c-Fos)in a concentration-dependent manner.This study also reaffirms the inhibitory effect of Helenalin on the non-classical NF-?B signalling pathway.Part ?: Investigating the effects of Helenalin on inflammatory bone destruction based on an LPS-induced cranial bone injury model and a type II collagen-induced RA animal modelWhile the classical NF-?B signalling pathway has been reported to affect bone metabolism in both physiological and pathological conditions,the non-classical NF-?B pathway has no effect on osteoclastogenesis in the basal state.However,it is required for excessive osteoclast formation in osteolytic diseases(caused by inflammation,infection,etc.).Therefore,the non-classical NF-?B pathway is more suitable as a target for the treatment of osteolytic diseases.In this thesis,two classical models of inflammatory bone destruction were developed to define further the inhibitory effects of Helenalin on osteoclastogenesis and function at the animal level.(I)Inhibitory effects of Helenalin on LPS-induced cranial bone injury in mice.LPS(5mg/kg)was injected subcutaneously into the cranial periosteum in the form of alternate day injections,and the cranial bone injury model was established after four injections.The experiment was divided into the Sham group,LPS+Vehicle group and LPS+Helenalin(1mg/kg)group;each group consisted of 5 mice.A Micro-CT scan and 3D reconstruction observed bone erosion.The results showed that compared to the Sham group,the LPS+Vehicle group could observe significantly denser bone traps and a significant decrease in bone mineral density(BMD)and bone volume fraction(BV/TV).In contrast,the LPS-induced osteolysis was effectively improved by Helenalin treatment,as evidenced by a reduction in surface traps and an increase in BMD and BV/TV.The results showed that LPS induced inflammatory cell infiltration,a significant increase in the percentage of bone erosion area,an increase in osteoclast production and a significant upregulation of osteoclast surface area to bone surface area(Oc S/BS).Helenalin administration significantly inhibited these pathological changes.ELISA measured the expression of RANKL in serum,and Helenalin significantly inhibited the upregulation of RNAKL in the Vehicle group.(II)Inhibitory effects of Helenalin on type II collagen-induced RA animal modelsThe CIA model in DBA1 mice was established by twice immunizing with type II collagen and complete Frey's adjuvant emulsion.The experiments were divided into standard control,CIA+Vehicle and CIA+Helenalin groups,with five mice in each group.Helenalin(1 mg/kg)was administered intraperitoneally as an alternate day dose and taken for analysis 22 days after administration.Arthritis index scores and paw volume measurements showed that Helenalin significantly inhibited the onset and progression of CIA in the mice;Micro-CT 3D reconstruction showed that Helenalin alleviated bone destruction in the ankle joint of CIA mice,as evidenced by reduced bone traps and increased BMD and BV/TV;pathological sections and scores showed that Helenalin significantly inhibited inflammatory infiltration,percentage of bone erosion area and Oc S/BS in the ankle joint of CIA mice,and Helenalin significantly inhibited the overproduction of osteoclasts in the ankle joint of CIA mice;ELISA results showed that Helenalin inhibited the expression of inflammatory factors in the ankle joint homogenates of CIA mice,and The ELISA results showed that Helenalin inhibited the expression of inflammatory factors in the ankle homogenates of CIA mice,and also suppressed the serum levels of RANKL and Anti-CII antibodies.Conclusion:Helenalin inhibited the expression and transcriptional activity of its downstream transcription factor NFATc1 by suppressing the non-classical NF-?B signalling pathway(mainly inhibiting the activity and protein level of NIK,the phosphorylation level of IKK?,p100 to p52 processing and Rel B/p52 nuclear translocation),which in turn downregulated osteoclast marker gene expression and ultimately inhibited osteoclast production and activation,alleviating inflammatory bone destruction in cranial injury models and CIA models.