Study On The Biological Connotation Of The Dynamic Evolution Of Blood Stasis Syndrome In Non-traumatic Femoral Head Necrosis Based On Syndrome And Symptom Combinatio

Background:Non-traumatic osteonecrosis of the femoral head(NONFH)is a common orthopedic disease that is prone to occur in young and middle-aged people.However,the non-surgical treatment options for early intervention are very limited,there are few targeted drμg s in pharmacological therapy,so total hip arthroplasty have to be the final choice for patients.The disease belong to the categories of "Bi syndrome" and "Gushi" in traditional Chinese medicine.Our previous researches has confirmed that "blood stasis" is the key pathogenesis running throμg h the whole course of NONFH.Moreover,with the development of the disease,it shows the characteristics that change from mild to severe,and then from severe to mild.The syndrome differentiation and treatment system of the early,middle and late stage has been established,but the biological connotation of the dynamic evolution process of the syndrome has not been revealed.Object:1.A cross-sectional study was designed to explore the characteristics of patients with different syndrome types under the guidance of the theory of "combination of syndrome and symptom",,so as to provide the basis for the follow-up study.2.To explore dynamic biomarkers to monitor NONFH blood stasis syndrome staging by dividing NONFH patients into early,middle and late stages,extensively collected their clinical symptoms under the guidance of the theory of "combination of syndrome and symptom",and formed a "disease-syndrome-symptom-gene" network fusion-based analysis by combing the NONFH-related expression profiling,clinical phenomics data and clinical validation.3.To evaluate pathological indicators and expression of the above candidate biomarkers with good clinical syndrome differentiation efficacy by establishing steroid-induced NONFH rat model,and further clarify the dynamic syndrome evolution characteristics of the model.Methods:1.A total of 80 NONFH patients were selected,consisted of early-stage(n=35),middle-stage(n=30)and late-stage(n=15).The gender,age,complain,TCM syndrome,hormone history or alcohol consumption history,imaging resμLts,blood lipids,coagμLation,platelet parameters and thrombosis were collected,and evaluate Harris hip joint score,clinical score and image score.2.A total of 84 steroid-induced NONFH patients and 20 non-NONFH patients were enrolled and randomly divided into a discovery cohort and a validation cohort.Peripheral blood samples from the discovery cohort were collected for transcriptomic detection and differentially expressed genes(DEGs)among three staging groups of NONFH were screened.Then,clinical symptoms of NONFH and the related genes were collected from HPO and DisGeNET.The "syndrome-symptom" interaction networks among the DEGs and the symptom-related genes of each NONFH stage were constructed and the candidate biomarkers for each stage were identified according to the network topological features,the co-expression correlation and network distance between DEGs and symptom-related genes.After that,real-time quantitative PCR(RT-PCR)was performed to verify the expression patterns of candidate biomarkers for each stage based on the validation cohort and their diagnostic efficacy was comprehensively evaluated.David bioinformatics resources 6.8 database for functional enrichment analysis3.A total of 20 healthy male SPF SD rats were randomly divided into four groups:SONFH-4w group,Control-4w group,SONFH-16w group and Control-16w group.For SONFH model groups,lipopolysaccharide(LPS)20 μg/kg,d-1 was injected on 1-2 day and methylprednisolone sodium succinate 40 mg/kg,d-1 was injected on 3-5 day;for the Control groups,normal saline was injected;penicillin sodium to prevent infection.The body weight,50%paw withdrawal threshold(50%MWT),macroscopic changes of femoral head,inflammatory factors,blood lipid,platelet function,coagμLation and bone metabolism indexes of the rats were collected.Hematoxylin and eosin(HE)staining and tartrate-resistant acid phosphatase(Trap)staining were used to observe the pathological changes of femoral head.RT-PCR and immunohistochemistry(IHC)staining were used to verify the expression levels of candidate biomarkers in peripheral blood and femoral head,respectively.Result:1.Among the 80 patients,58 were steroid-induced(72.50%),4 alcohol-induced(5.00%),and 18 idiopathic(22.50%),with the age of 39.54 ± 13.89 years old and the course of disease of 21.52 ±35.39 months.143 hips were involved totally as 63 cases(78.75%)occurred in unilateral hip and 17 cases occurred(21.25%)bilateral hips.Most patients at the early-stage group were in ARCO Ⅰ or Ⅱ stage,most patients at the middle-stage were in ARCO Ⅱ and Ⅲ stage,and most patients at the late-stage were in ARCO Ⅲ and Ⅳ stages.The Harris score and joint range of motion were significantly different among the three groups,and the score in the early-stage group was significantly higher than that in the late-stage group(all P<0.05).The clinical core outcome set was significantly lower in the early-stage group than in the middle-stage group and late-stage group;The VAS score of the early-stage group was significantly lower than that the middle-stage group,range of motion of hip flexion score of the early-stage group was significantly lower than the middle-stage group and late-stage group,the walking distance score of the early-stage group was significantly lower than the middle-stage group(all P<0.05).As for radiographic outcomes,the total scores of the early-stage group was significantly lower than the middle-stage group and the late-stage group,the middle-stage group was lower than the late-stage group,too;there were differences among the three groups in femoral head morphology score and osteoarthritis score(P<0.05).The frequency of "hip joint pain" and "triggered by exercise" in each NONFH groups was more than 90%;the frequency of "tenderness" was more than 70%,with late-stage as the highest.The frequency of "limitation of joint mobility" in the middle-stage and late-stage group was higher."Obesity "appeared more frequently in the early-stage group;"intolerance of cold in lower limbs" and "leg stiffness" appeared more frequently in the middle-stage group;"claudication","knee pain","intolerance of cold in lower limbs","tongue petechia","joint swelling","waist soreness","lower limb weakness",and "fatigable weakness" appeared more frequently in the late-stage group.In terms of biochemical indicators,the total cholesterol(TC)of the middle-stage group was significantly lower than that in the late-stage group,and the low density lipoprotein(LDL)of the early-stage group and middle-stage group were lower than that the late-stage group.Compared with the middle-stage group and the late-stage group,the thrombomodμLin(TM)of early-stage group had significant effects(all P<0.05).2.The early-stage-related hubs were associated with the typical clinical symptoms of NONFH at the early stage,such as "episodic pain","hip pain",etc..The corresponding clinical symptoms of the mid-stage-related hubs were involved into "hip pain","headache",etc..In contrast,the late-stage-related hubs were distinctly associated with several typical symptoms like "hip pain","night sweats",etc..Functionally,the different stage-related hubs were enriched into various pathways related to the main pathological changes during the progression of NONFH,such as PI3K-Akt signaling pathway,Chemokine signaling pathway and Apoptosis.Among them,6 genes of early-stage,13 genes of mid-stage and 6 genes of late-stage were selected as candidate biomarkers,respectively,for the RT-PCR validation.Of note,the expression levels of PRKCA,CD4 and PIK3CD were all dramatically up-regμLated in the early-stage NONFH patients(all P<0.05)and had a good performance for screening the early-stage NONFH patients from the non-NONFH and other stage groups.Similarly,the elevated expression of STAT2,CXCR4 and LPAR1 were all distinguish features of the late-stage NONFH patients.3.The body weight of rats in the SONFH group increased slowly significantly at 1w,2w and 3w,and the 50%MWT was significantly decreased at 1w-7w,9w and 10w(all P<0.05).In the SONFH group,red blood stasis was observed under the macroscopic cartilage of the femoral head.HE staining showed that the relative area ratio of trabecμLar bone in the SONFH-4w group was lower than that in the Control-4w group,that in the SONFH-16w group was lower than that in the Control-16w group,and that in the SONFH-4w group was higher than that in the Control-4w group and that in the SONFH-16w group was higher than that in the Control-16w group(all P<0.05).The positive area of Trap staining in the SONFH-4w group was significantly larger than that in the Control-4w group,tt coμLd be observed in the SONFH-16w group but rarely in the Control-16w group.The levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were significantly increased in the SONFH-4w group and SONFH-16w group,while the levels of TC,TG and LDL-C were significantly increased in the SONFH-4w and SONFH-16w groups,but HDL-C was significantly decreased.The levels of thromboxane B2(TXB2)and 6ketoprostaglandin F1 alpha(6-keto-pgFl alpha)were significantly increased in the SONFH-4w group and SONFH-16w group,while the activated partial thromboplastin time(APTT)and prothrombin time(PT)were significantly decreased and thrombin time(TT)was significantly increased in the SONFH-4w group and SONFH-16w group.The levels of nuclear factor κB receptor activating factor ligand(RANKL)and osteoprotegerin(OPG)were significantly increased in the SONFH-4w group and SONFH-16w group,while the RANKL/OPG ratio in the SONFH-16w group was increased(all P<0.05).Regarding candidate biomarkers,early candidate markers PRKCA,PIK3CD,and CD4 were significantly overexpressed in the SONFH-4w group(P<0.05),while late candidate markers STAT2,CXCR4,and LPAR1 were significantly overexpressed in the SONFH-16w group(P<0.05).p-PRKCA and PIK3CD were positively expressed in bone of SONFH-4w group,and CD4 was positively high expressed in SONFH-4w group.In the late-stage,p-STAT2 and LPAR1 showed the positive staining in the bone of femoral head in SONFH-16w group,while CXCR4 showed positively high expressed in SONFH-16w group.Conclusion:1.The main symptoms and secondary symptoms of patients with different syndromes of NONFH blood stasis syndrome are different.The Harris score,clinical core outcome set and radiographic Outcomes score of the early-stage are better than those of the middle or late stages.2.The biological connotation of NONFH blood stasis syndrome in different stages has different focuses.A list of novel candidate dynamic biomarkers to monitor NONFH staging were identified,among which six candidate genes show a good performance for screening the early-and late-stage NONFH patients respectively,including PRKCA,CD4,PIK3CD,STAT2,CXCR4 and LPAR1.3.The high expressions of PRKCA,CD4,PIK3CD,STAT2,CXCR4 and LPAR1 may be highly correlated with the dynamic evolution of the pathogenesis of NONFH blood stasis syndrome.