| Objective(1)To investigate the presence of biological rhythm disorders in patients with non-traumatic osteonecrosis of the femoral head(ONFH)and to examine the expression of circadian locomotor output cycles(CLOCK)and period circadian regulator 1(PER1)in blood and femoral head specimens from patients with steroid-induced osteonecrosis of the femoral head(SONFH).(2)To employ network pharmacology analysis methods to identify potential active components of Huoxue Tongluo prescription and their corresponding action targets,construct and analyze the Huoxue Tongluo prescription active component-target network,select the relevant targets for steroid-induced osteonecrosis of the femoral head,and validate the findings using a rat model of SONFH.(3)To observe the effects of various concentrations of serum containing Huoxue Tongluo prescription on human bone marrow mesenchymal stem cells(hBMSCs)Bmall,osteogenic genes,and Wnt/β-catenin signaling pathway expression,and investigate the mechanism by which Huoxue Tongluo prescription promotes osteogenic differentiation of hBMSCs.Methods(1)Non-traumatic ONFH patients treated conservatively at Baoji Traditional Chinese Medicine Hospital from January to December 2020 were included in the study.The presence of circadian disturbances in patients and healthy controls was compared using the Pittsburgh Sleep Quality Index,Life Satisfaction Rating Scale,and Symptom Checklist.Additionally,20 patients with SONFH and 10 healthy volunteers were chosen to assess the expression of biological clock genes at different time points.Peripheral venous blood(approximately 15 mL)was collected from both groups at 06:00,12:00,and 18:00 on the same day.Enzyme-linked immunosorbent assay(ELISA)was utilized to detect CLOCK,Bmal1,and PER1 genes in plasma.Furthermore,immunohistochemical methods were employed to determine the expression of CLOCK,Bmal1,and PER1 genes in the necrotic and normal regions of the femoral head using six femoral head specimens from ONFH patients who underwent joint replacement surgery.(2)Utilizing network pharmacology methods,effective active components and targets of Huoxue Tongluo prescription were screened,SONFH targets were obtained,and common targets between the active components of Huoxue Tongluo prescription and SONFH were identified.A protein-protein interaction(PPI)network was constructed,followed by enrichment analysis of biological functions and signaling pathways,and further molecular docking verification.Moreover,a SONFH rat model was established and divided into control,model,and Huoxue Tongluo prescription groups.Trabecular number(Tb.N),bone volume/total volume(BV/TV),trabecular thickness(Tb.Th),and trabecular separation(Tb.SP)were analyzed in the femoral heads of each group.Hematoxylin and eosin(HE)staining determined the rate of empty bone lacunae in rats from each group.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)detected alkaline phosphatase(ALP)and Bmall gene mRNA expression in rat femoral heads.Immunohistochemical staining assessed ALP and Bmall protein expression in rat femoral heads.(3)Employing human bone marrow mesenchymal stem cells(hBMSCs)as the research vector,hBMSCs were infected with Bmal1 overexpression and silencing lentivirus(groups:NC lentivirus,Bmall overexpression lentivirus,NC shRNA lentivirus,Bmall shRNA silencing lentivirus).Osteogenic-related gene expression,such as Runx2 and BMP2,was examined using RT-qPCR and Western blot methods.Concurrently,the effects of Bmall expression and silencing on the Wnt/β-catenin signaling pathway were observed.Additionally,serum containing Huoxue Tongluo prescription at various concentrations was prepared,and low,medium,and high concentrations of drug-containing serum were designed to induce osteogenic differentiation of hBMSCs treated with high concentrations of dexamethasone(Dex)(groups:model group,low concentration drug-containing serum,medium concentration drug-containing serum,high concentration drug-containing serum,high concentration drug-containing serum+silencing Bmall group).Alkaline phosphatase(ALP)staining and Alizarin Red staining were utilized to observe the effects of drug-containing serum on hBMSC osteogenic differentiation.RT-qPCR and Western blot techniques examined the impact of low,medium,and high concentrations of drug-containing serum on hBMSC Bmall expression,osteogenic-related genes,and the Wnt/β-catenin signaling pathway.Results(1)A total of 159 non-traumatic ONFH patients were included,comprising 59 SONFH patients,42 alcoholic ONFH patients,and 58 idiopathic ONFH patients.Additionally,57 healthy volunteers were recruited.No significant difference in age and gender was observed between the non-traumatic ONFH group and the healthy control group(P>0.05).Non-traumatic ONFH patients exhibited poorer sleep and life satisfaction compared to healthy individuals.The Pittsburgh sleep quality index in the non-traumatic ONFH group was significantly higher than that in the healthy control group(P<0.05).Furthermore,life satisfaction scores in the non-traumatic ONFH group were significantly lower than those in the healthy control group(P<0.05),while the symptom checklist scores for the non-traumatic ONFH group were significantly higher than those of the healthy control group(P<0.05).The life satisfaction score in the SONFH group was significantly lower than that in the alcoholic ONFH group and idiopathic ONFH group(P<0.05).Moreover,the Pittsburgh sleep quality index and life satisfaction scores in the kidney deficiency and blood stasis group were significantly lower than those in the phlegm stasis accumulation group and the qi stagnation and blood stasis group(P<0.05).ELISA detection revealed no significant difference in CLOCK protein expression between the healthy control group and the SONFH group at 06:00 am(P>0.05).However,CLOCK and Bmall protein expression in the healthy control group at 12:00 pm and 18:00 pm was higher than that in the SONFH group(P<0.05).PER1 protein expression in the SONFH group at 06:00 am and 12:00 noon was significantly higher than that in the healthy control group(P<0.05).Immunohistochemical results demonstrated that protein expressions of CLOCK and Bmall,the core genes of the biological clock in necrotic bone tissue,were significantly lower than those in normal bone tissue(P<0.05),while PER1 protein expression in necrotic bone tissue was significantly higher than that in normal bone tissue(P<0.05).(2)Analysis revealed 135 effective active ingredients in the Huoxue Tongluo prescription,with 17 common ingredients among its constituent drugs.Utilizing SwissTargetPrediction,160 drug targets were obtained after prediction,combination,and weight removal.An interaction network of the Huoxue Tongluo prescription active ingredient targets was constructed using Cytascape 3.7.2 software,resulting in a total of 309 nodes and 1859 edges.Ninety-one potential targets were identified for the treatment of SONFH with the Huoxue Tongluo prescription.GO analysis of these 91 potential target genes demonstrated their main enrichment in response to steroid hormones,cell response to chemical stress,response to oxidative stress,regulation of metal ions,drugs,DNA binding transcription factor activity,and regulation of reactive oxygen species metabolism processes.KEGG pathway enrichment analysis indicated a primary concentration on lipid and atherosclerosis,prostate cancer,chemical carcinogenesis receptor activation,hepatitis B,fluid shear stress,and TNF signaling pathway.The rate of empty bone lacunae in the femoral head of rats in the model group was significantly higher than that in the control group and the Huoxue Tongluo prescription group.The BV/TV and Tb.N of the femoral head in the model group were significantly lower than those in the control group and the Huoxue Tongluo prescription group(P<0.05).The expression of ALP gene mRNA in the bone tissue of rats in the model group was significantly lower than that in the control group and the Huoxue Tongluo prescription group(P<0.05).Additionally,the expression of ALP gene mRNA in the bone tissue of rats in the control group was significantly higher than that in the Huoxue Tongluo prescription group(P<0.05).The expression of Bmal1 gene mRNA in both the Huoxue Tongluo prescription group and the model group was significantly lower than that in the control group(P<0.05).The immunohistochemical mean optical density values of ALP and Bmall in the control group were significantly higher than those in the model group and the Huoxue Tongluo prescription group(P<0.05).(3)RT-qPCR and Western blot results showed that the expression of osteogenic-related genes such as Runx2 and BMP2 in the overexpression Bmall lentivirus group was significantly higher than in the NC lentivirus group(P<0.05).In contrast,the expression of Runx2 and BMP2 in the Bmall shRNA silenced lentivirus group was significantly lower than in the NC shRNA silenced lentivirus group(P<0.05).The Bmall overexpression lentivirus group had significantly increased expression of Wnt2 and β-Catenin compared to the NC lentivirus group(P<0.05),while the Bmall shRNA silenced lentivirus group had significantly reduced expression of Wnt2 and β-Catenin compared to the NC-shRNA silenced lentivirus group(P<0.05).Additionally,low,medium,and high concentrations of drug-containing serum enhanced alizarin red and ALP staining of hBMSCs.RT-qPCR and Western blot results showed that,compared to the model group,the intervention of serum containing different concentrations of Huoxue Tongluo prescription significantly upregulated the expression of Bmall,BMP2,and Runx2 in hBMSCs(P<0.05)in a concentration-dependent manner.The expressions of Bmall,BMP2,and Runx2 in the high concentration drug-containing serum+silent Bmall group were significantly lower than in the high concentration drug-containing serum group(P<0.05).For the Wnt/β-Catenin signaling pathway,intervention with different concentrations of Huoxue Tongluo prescription-containing serum significantly upregulated Wnt2 and β-Catenin expression in a concentration-dependent manner(P<0.05).The expression of Wnt2 and β-Catenin in the high concentration drug-containing serum+silencing Bmall group was significantly lower than in the high concentration serum group(P<0.05).ConclusionNon-traumatic ONFH patients exhibit mild sleep disorders and disordered expression of biological clock genes.Bmall is expressed at low levels in the necrotic region of femoral head specimens from patients with SONFH.Overexpression of Bmall activates the Wnt/β-Catenin signaling pathway,promoting bone formation.The mechanism of Huoxue Tongluo prescription in treating SONFH may involve upregulation of Bmall expression,which in turn activates the Wnt/β-Catenin signaling pathway to promote bone formation. |
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