| Objective: avascular necrosis of the femoral head(afnh)is a common clinical disease with high disability rate and difficult treatment.Traumatic afnh is one of the most common types,and the incidence rate is gradually increasing.Surgical treatment of this disease has many disadvantages,such as large trauma,high medical cost,and poor long-term effect.Therefore,non-surgical treatment of afnh is an important clinical demand.Taohong Siwu Decoction is a classic blood activating prescription in traditional Chinese medicine.It has the function of promoting the repair of necrotic femoral head,improving the local blood supply of femoral head,and making the repair of necrotic femoral head;The mechanism may be related to promoting the expression of angiogenesis related factors such as VEGF.However,how Taohong Siwu Decoction regulates the expression of VEGF and other angiogenesis related factors,and the internal mechanism and key factors of promoting angiogenesis and repairing injured blood vessels have not been truly revealed and clarified.Exosome miR-126 is a key factor regulating angiogenesis,especially plays an important role in pathological angiogenesis and repair,and is an important target for treatment.The purpose of this study is to establish a model of traumatic femoral head necrosis and to clarify the therapeutic effect of Taohong Siwu Decoction on traumatic avascular necrosis of femoral head;Then potential therapeutic targets and pathways were screened by network pharmacology;From the perspective of angiogenesis,the molecular mechanism of Taohong Siwu Decoction in the treatment of traumatic femoral head necrosis was further clarified from the mir-126/vegf/notch signal axis,so as to provide a basis for clinical application.Methods:(1)the rat model of traumatic femoral head necrosis was established by simulating the pathological state of clinical traumatic femoral head necrosis.According to the random number table,they were divided into 3 groups: 12 in the normal group,12 in the model group and 12 in the sham operation group.Normal group: normal feeding without special intervention;In the model group,the round ligament was cut off and the basal periosteum of the femoral neck was stripped;The sham operation group was operated on,but the ligament was not cut and the joint capsule was not damaged.After the intervention,all the rats in the three groups were placed in a cage with sufficient space,and the rat food was placed above the cage.The lower limbs were required to bear weight when eating.After 8 weeks,the femoral head and femur were amputated.The rat model of traumatic femoral head necrosis was evaluated by micro CT examination,histomorphological observation and empty bone lacuna rate detection.(2)Taohong Siwu Decoction was used to intervene the rat model of traumatic avascular necrosis of femoral head in order to determine the therapeutic effect and optimal dose of Taohong Siwu Decoction.48 rats were adaptively fed for 1 week after purchase to adapt to the environment,and the experimental rats were observed to have no obvious diseases and to be healthy before grouping.According to the random number table,48 rats were divided into 6 groups:normal group,model group,model western medicine group(positive control group)and model Taohong Siwu Decoction group(low,medium and high Taohong Siwu Decoction groups),with 8 rats in each group.Normal group: the rats were given routine feeding.After the success of modeling in the model group and the model treatment group,normal saline was given by gavage;Model group: the rats in the model group were made with the method of the first part.After the model was successfully made,they were fed with conventional feed + normal saline by gavage;Model western medicine group: the rats in the model group were modeled by the first part of the method.After successful modeling,they were given conventional feed +alprostadil intravenous injection(intravenous injection 0.13 μ g/ time / day).Model treatment group: after successful modeling,the rats were randomly divided into three groups: low,medium and high Taohong Siwu Decoction,which were given Taohong Siwu Decoction by gavage and conventional feeding respectively.The rats in the 6groups were taken after 4 weeks of continuous intervention.The method of taking materials was the same as that in the first part.To evaluate the effect of Taohong Siwu Decoction in the treatment of femoral head from the aspects of gross morphology,histological morphology,empty bone lacuna rate,micro CT scanning and femoral head microvessel density.(3)Using network pharmacology to predict the material basis and target of Taohong Siwu Decoction for femoral head necrosis.The chemical components of Taohong Siwu Decoction and the corresponding targets were selected from tcmsp database;Then,the targets of active ingredients were predicted by universal protein(Uni Prot).Genecards database,OMIM database and Pharm GKB database were used to search for potential targets for the treatment of femoral head necrosis.By using string database and cytoscape3.8.0 software,the network of active components and key targets of Taohong Siwu Decoction was constructed.Through r4.0.2 software,the core gene of Taohong Siwu Decoction avascular necrosis of femoral head was enriched and analyzed by go gene function annotation BP,CC,MF(biological process,cell component,molecular function)and KEGG pathway.Finally,the molecular docking of the core target was carried out.(4)Based on the mir-126/vegf/notch signal axis,the molecular mechanism of Taohong Siwu Decoction in the treatment of traumatic femoral head necrosis was elucidated from the perspective of angiogenesis.After 32 rats were adaptively fed for 1W to adapt to the environment,they were randomly divided into 4 groups: normal group,model group,agomir-126 group(model agonist group)and Taohong Siwu Decoction group(model traditional Chinese medicine group),with 8 rats in each group;Except the normal group,the other three groups were modeled.Intervention method: the normal group and the model group were fed with conventional feed + normal saline by gavage;The model agonist group was given conventional feed +agomir-126 by tail vein injection(intravenous injection(20mg/kg)/ time)after successful modeling,and was injected 3times in 4W;The rats in Taohong Siwu Decoction group were given a high dose of Taohong Siwu Decoction 36g/kg by gavage after successful modeling,and the rest were fed the same as before for 4W.After 4 weeks of continuous intervention,rats were fasted for 12 hours,and blood samples were collected first;Then the femoral head was amputated.Serum exosomes were extracted by kit method,the content of miR-126 was detected by q PCR,the number of EPCs was identified by flow cytometry,the expression of VEGFA,Notch1,VEGFR-2 and Dll4 in femoral head was detected by WB,and the expression of VEGFA,Notch1,VEGFR-2 and Dll4 was detected by immunohistochemistry.Results:(1)from the aspect of appearance,there was no deformation and collapse of the femoral head in the normal group,and the surface of articular cartilage was smooth and glossy;In the sham operation group,the proximal surface of the articular cartilage of the femoral head was slightly dark,the gloss was acceptable,and there was no obvious deformation and collapse in the appearance;The articular cartilage surface of the femoral head in the model group was dark,lustrous,large in area,brittle in bone,and slightly sunken in some parts.Micro CT showed that the shape of femoral head in the normal group and the sham operation group was regular and smooth,and the femoral head bone in the model group was obviously damaged with collapse.He staining showed that in the normal group,the cartilage surface was smooth and complete,the morphology of chondrocytes was normal,and there were a few empty bone lacunae;The sham operation group was almost the same as the normal group;In the model group,there were inflammatory cell infiltration,and the deep chondrocyte fossa was enlarged and stained shallowly.(2)Micro CT three-dimensional reconstruction showed that the femoral head in the normal group had good shape,smooth and no obvious collapse;In the model group,obvious destruction of the femoral head bone with obvious collapse was observed;In the model western medicine group,the femoral head was slightly less smooth,with a small amount of necrosis and deformation;In the low dose group,the femoral head bone was partially destroyed and partially collapsed;In the middle dose group,the shape of femoral head was slightly changed,slightly rough,and a small amount of osteonecrosis and collapse were observed;In the high-dose group,the morphology of the femoral head was slightly changed,the femoral head was slightly less smooth,a small amount of necrosis,and slightly deformed.The microvessel density of the femoral head showed that in the normal group,the femoral head structure was clear and the capillaries were evenly distributed;In the model group,the local structure of femoral head was disordered,inflammatory cells infiltrated,and a small amount of capillaries grew;In the model western medicine group,a moderate amount of capillaries grew and partially extended to the necrotic area;In the low dose group,some capillaries grew and a small amount extended to the necrotic area;In the middle dose group,more capillaries grew and extended to the necrotic area;In the high-dose group,a large number of capillaries were seen,some of them gathered and extended to the necrotic area.(3)Through tcmsp,genecards,OMIM,Pharm GKB,string,Pub Chem and other databases,a total of 130 active ingredients and 210 effective targets of Taohong Siwu Decoction were obtained;81 potential targets of Taohong Siwu Decoction for the treatment of avascular necrosis of femoral head were obtained.Among them,Taohong Siwu Decoction has 15 potential core targets for the treatment of avascular necrosis of the femoral head,including IL6,VEGFA,AKT1,1l1 b,TP53,EGF,PPARG,Jun,MMP9,CASP3,HIF1 A,PTGS2,CXCL8,EGFR and CCL2.147 potential pathways were found,including AGE-RAGE signaling pathway,HIF-1signaling pathway,VEGF signaling pathway,MAPK signaling pathway,IL-17 signaling pathway and PI3 K Akt signaling pathway.Molecular docking results also showed that VEGFA had strong biological activity.(4)Serum exosome miR-126content: compared with the normal group,the exosome miR-126 content in the agonist group was significantly increased;Compared with the model group,the contents of miR-126 in exosomes of the other three groups were significantly higher;The content of agonist group was slightly higher than that of Taohong Siwu Decoction group,but the difference was not statistically significant.The number of EPCs in peripheral blood: the normal group had the highest content;The content of agonist group was the same as that of Taohong Siwu Decoction group,but it was significantly higher than that of model group,and the difference was statistically significant.WB detection of VEGFA content in rat femoral head: compared with the model group,VEGFA content in the other three groups was higher,especially in the normal group;However,there was no significant difference among the other three groups.VEGFR2content: compared with the model group,the VEGFR2 content of the other three groups increased,and the difference was statistically significant;There was no significant difference among the other three groups.Notch1 content: compared with the model group and the normal group,the Notch1 content in the agonist group and the Taohong Siwu Decoction group increased,and the difference was statistically significant;There was no significant difference among other groups.Dll4 content:compared with the normal group and the model group,the Dll4 content in Taohong Siwu Decoction group increased,and the difference was statistically significant;There was no significant difference between the other groups.Conclusion:(1)the rat model of traumatic femoral head necrosis can be effectively established by surgical modeling,which can be used to simulate the mechanism of clinical traumatic femoral head necrosis.(2)The middle and high doses of Taohong Siwu Decoction can improve the necrosis of rat femoral head,promote local angiogenesis,and alleviate the process of rat femoral head necrosis.The high dose group has the best effect.(3)According to the network pharmacology,it is predicted that the core target genes of Taohong Siwu Decoction for the treatment of femoral head necrosis include IL6,VEGFA,AKT1,1l1 b,etc.,and the core target VEGFA has strong activity.The closely related pathways are AGE-RAGE signal pathway,HIF-1signal pathway,VEGF signal pathway,MAPK signal pathway,etc.(4)The mechanism of Taohong Siwu Decoction in treating femoral head necrosis is related to its promotion of angiogenesis.Its molecular mechanism may be that it can up regulate the expression of serum exosome miR-126,activate VEGF signal to unblock collaterals,promote endothelial cell proliferation,and thus make local angiogenesis. |
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