| BackgroundOsteoporosis is a common metabolic bone disease in middle-aged and elderly people,characterized by an imbalance in bone metabolism leading to changes in bone microstructure,decreased bone mass,increased bone fragility,and increased risk of fractures,which is one of the important causes of death in the elderly.Osteoporosis has become a significant global public health issue,and fractures caused by osteoporosis require a significant amount of medical resources,imposing a heavy burden on society,families,and the economy.Currently,the clinically used anti-osteoporotic drugs still have many side effects,such as gastrointestinal symptoms,osteonecrosis,and atypical fractures.Therefore,the search for safe and effective drugs has been a hot topic in osteoporosis research.Isoliquiritin is a small molecule flavonoid drug.Previous literature has shown that it has various effects,such as regulating glucose and lipid metabolism,inhibiting inflammation,and anti-apoptosis.However,its effects on osteoclasts have not been studied.Therefore,in this study,in vivo and in vitro experiments were conducted to explore the effects of isoliquiritin on osteoclasts and to investigate its biological mechanisms,providing a theoretical basis for clinical translational applications.ObjectiveThe aim of this study is to investigate the effects of the natural small molecule compound,isoliquiritin,on osteoclast differentiation and bone resorption function,to clarify its therapeutic effects on osteoporosis,and further explore its regulatory mechanisms.The goal is to provide new approaches for the prevention and treatment of osteoporosis.MethodsPart Ⅰ: Effects of Isoliquiritin on Osteoclast Differentiation and Function(1)In vitro experimental model of osteoclasts was established.Six-week-old male C57BL/6 femurs were obtained and the bone marrow cells were flushed with M-CSF at a concentration of 30ug/ml to induce the differentiation of primary bone marrow macrophages(BMDM).RANKL at a concentration of 100ug/ml was then used to stimulate BMDM to differentiate into osteoclasts,creating an in vitro experimental model.(2)CCK-8 assay was used to evaluate the cytotoxic effect of isoliquiritin on osteoclasts,and an appropriate cell drug concentration was selected.(3)Different concentrations of isoliquiritin were used to intervene in the process of osteoclast differentiation.TRAP staining was used to observe osteoclast differentiation,and Western Blot,RT-q PCR,and other methods were used to detect the expression of bone resorption-related genes and protein function to validate the effects of isoliquiritin on osteoclast differentiation and function.Part Ⅱ: Effect of isoliquiritin on osteoporosis in ovariectomized mice(1)An in vivo experiment was conducted to establish an osteoporosis model.Eight-week-old female C57BL/6 mice were randomly divided into four groups: sham group(Sham),ovariectomy group(OVX),low-dose isoliquiritin group(ISL-LD),and high-dose isoliquiritin group(ISL-HD).The Sham group underwent sham surgery only,while the OVX,ISL-LD,and ISL-HD groups underwent ovariectomy to establish the osteoporosis model.(2)After successfully establishing the osteoporosis model through ovariectomy,the Sham and OVX groups received intragastric injections of PBS,while the ISL-LD and ISL-HD groups received intragastric injections of isoliquiritin at doses of 20 mg/kg and 50mg/kg,respectively,for 6 weeks.(3)After the treatment period,femoral specimens were taken for micro-CT,H&E staining,and TRAP staining to compare bone density,trabecular thickness,trabecular number,and osteoclast number.The protective effect of isoliquiritin on bone loss induced by ovariectomy-induced osteoporosis was validated at the animal model level.Part Ⅲ: Investigation of the Mechanisms by which Isoliquiritin Affects Osteoclast Differentiation and Function(1)Osteoclast models were constructed and treated with different concentrations of isoliquiritin.The expression of glycolysis-related proteins in osteoclasts was examined using Western Blot and other methods to validate the effects of isoliquiritin on the glycolytic metabolism process during osteoclast differentiation.(2)The 3D structure of isoliquiritin was downloaded from Pub Chem data and docked with glycolysis-related proteins using Auto Dock.The interaction patterns were analyzed to predict the specific targets of isoliquiritin in affecting the glycolytic process.(3)RAW264.7 cells were cultured and pre-treated with different concentrations of isoliquiritin.An inflammation model was induced by LPS stimulation,and the expression of inflammation-related genes and proteins were examined using Western Blot and RT-q PCR to validate the inhibitory effects of isoliquiritin on inflammation.(4)Macrophages were pre-treated with different concentrations of isoliquiritin and stimulated with LPS to collect the supernatant.The collected supernatant was co-induced with RANKL to differentiate BMDM into osteoclasts.The differentiation and expression of osteoclast-related proteins were examined using TRAP staining,Western Blot,and other methods to validate the effects of isoliquiritin on osteoclast differentiation and function by affecting the inflammatory microenvironment.Results1.CCK-8 assay results showed that isoliquiritin at concentrations of 160 u M and below did not exhibit significant cytotoxicity towards BMDMs and had no significant effect on their cell viability.Therefore,in this study,we ultimately chose 20 u M and 40 u M as the drug doses for stimulating cells.2.TRAP staining showed that isoliquiritin significantly inhibited the differentiation of macrophages into osteoclasts.Compared with the control group,the number and volume of osteoclasts stained in the experimental group were significantly reduced.Quantitative analysis of osteoclast number and area revealed that 40 u M isoliquiritin had a stronger inhibitory effect on osteoclast differentiation than 20 u M.3.Results from Western blotting and RT-q PCR indicated that during the process of RANKL-induced differentiation of BMDMs into osteoclasts,the expression of osteoclast-related genes and proteins,such as NFATc1,CTSK,ACP5,and MMP9,was significantly increased.After treatment with isoliquiritin,the expression of these osteoclast-related genes and proteins in the experimental group was downregulated compared to the control group.Quantitative analysis revealed that 20 u M and 40 u M isoliquiritin had a significant inhibitory effect on the expression of osteoclast-related genes and proteins,and 40 u M had a stronger inhibitory effect.4.The significant protective effect of isoliquiritin on bone loss was observed in mice with ovariectomy-induced osteoporosis.Micro-CT analysis revealed that OVX mice exhibited evident bone loss after 6 weeks,as evidenced by a decrease in bone density,cortical thickness,bone volume fraction,trabecular number,and trabecular thickness,along with an increase in trabecular separation.In contrast,mice treated with isoliquiritin showed a reversal in these bone parameters.Furthermore,H&E and TRAP staining showed a marked increase in osteoclasts surrounding the trabecular bone in the OVX group,which was significantly reduced in the isoliquiritin treatment group,particularly in the ISL-HD group.5.Western blot analysis revealed that the expression of glycolytic proteins,including HK1,PFK,PKM,and LDHA,was significantly upregulated during RANKL-induced osteoclast differentiation in RAW264.7 cells.However,the expression of these proteins was reduced to varying degrees after treatment with isoliquiritin.Quantitative analysis showed that high concentrations of isoliquiritin strongly inhibited the expression of glycolytic proteins in osteoclasts.Additionally,molecular docking studies revealed that isoliquiritin bound to AKR1A1 through hydrogen bonding and hydrophobic interactions,with strong binding affinity.Docking mode analysis suggested that AKR1A1 may be a potential drug target for the pharmacological action of isoliquiritin.6.In the LPS-induced inflammation model of RAW264.7 macrophages,the expression of inflammation-related genes was inhibited by pretreatment with isoliquiritin.q PCR results showed that the expression of TNF-α,IL-1β,IL-6,and IL-10,which are related to inflammation,were significantly upregulated after LPS stimulation.However,the upregulation of inflammation-related genes was inhibited by pretreatment with isoliquiritin.Western blot results showed that compared with the control group,the expression of inflammation-related proteins in macrophages stimulated by LPS was significantly decreased after pretreatment with isoliquiritin,which was consistent with the trend of RNA expression mentioned before.7.After pretreatment with isoliquiritin and stimulation with LPS,the supernatant was collected and used to induce osteoclast differentiation with RANKL.The TRAP staining results showed that the ability of the supernatant pretreated with isoliquiritin to induce osteoclast differentiation was significantly weakened compared with the control group,and the number and volume of osteoclasts produced were significantly reduced.At the same time,Western blot results showed that the supernatant pretreated with isoliquiritin inhibited the bone resorption function of osteoclasts,hindered the expression of osteoclast-related proteins,and the inhibitory effect was stronger in the high-dose isoliquiritin-treated group.ConclusionOn the one hand,isoliquiritin inhibits the glycolysis process of osteoclasts,while on the other hand,it impedes osteoclast differentiation and bone resorption function by suppressing inflammatory responses and improving the inflammatory microenvironment,thereby alleviating OVX-induced osteoporosis.Moreover,isoliquiritin may exert its inhibitory effect on glycolysis by binding with AKR1A1.In summary,isoliquiritin may be a potential drug for the treatment of osteoporosis. |
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