Regulation of Quan Du Zhong capsule on VEGF/bFGF and expression of Bcl-2/Bax and Caspase-3 protein in the repairing process of canine femoral head necrosis

SUN Rui-yan, YIN Peng-kai, WEI Wei, DENG Xiao-lei, HOU De-cai

1.Liaoning University of Traditional Chinese Medicine, Shenyang 110000, China

2.Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110000, China

3.Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110000, China

Keywords:

ABSTRACT Objective: To explore the repair and treatment effect of Quan Du Zhong capsule on necrosis of femoral head in dogs.Methods:Totally 12 beagles were randomly divided into normal group,model group, Quan Du Zhong capsule group and Xianlinggubao capsule group, with three in each group.In addition to the normal group, the other groups established the femoral head necrosis model by liquid nitrogen alternative freezing.The normal group and the model group did not have any intervention during the modeling period, and the Quan Du Zhong group began to receive the Quan Du Zhong by gavage on the day of modeling; Xianlinggubao capsule group was given Xianlinggubao capsule by gavage once a day for 12 consecutive weeks on the day of modeling.The levels of VEGF and bFGF in the blood vessels of each group at the 12th week were compared, and the ratios of BMD, BS/BV, BV/TV, Tb.Th, Tb.N were measured by Micro CT, and the expressions of Bcl-2, Bax, Caspase-3 proteins were detected by immune reaction.Results: 1.Compared with the normal group, the level of serum VEGF and bFGF in the model group decreased after 12 weeks of modeling (P＜0.05); Compared with the model group, the levels of serum VEGF and bFGF water in the Xianlinggubao capsule group and the Quan Du Zhong capsule group increased on average at the 12th week of modeling, with statistical difference (P＜0.05).The level of the Quan Du Zhong capsule group was the highest,followed by the Xianlinggubao capsule group.2.Compared with the normal group, BMD,BS/BV, BV/TV, Tb.Th and Tb.N in the model group were lower, and Tb.SP were higher, the results were statistically significant (P＜0.05).Compared with the model group, the BMD, BV/TV, Tb.Th and Tb.N of Xianlinggubao capsule group and the total eucommia capsule group increased,while the BS/BV and Tb.SP decreased (P＜0.05).3.The Quan Du Zhong capsule group and Xianlinggubao capsule group could significantly increase the expression of bcl-2 protein in the femoral head of dogs, which was significantly different from the model group (P＜0.05).The expression of bax protein in the femoral head of dogs in the Quan Du Zhong capsule group and the Xianlinggubao capsule group was significantly reduced compared with the model group (P＜0.05).The expression of caspase-3 protein in the femoral head of dogs was significantly reduced in the Quan Du Zhong capsule group and Xianlinggubao capsule group,which was significantly different from the model group (P＜0.05).Conclusion: Quan Du Zhong capsule can increase the expression of VEGF and bFGF in serum, increase the expression of bcl-2,inhibit the expression of bax, and reduce the expression of caspase-3, which plays a synergistic role in the treatment of avascular necrosis of the femoral head, and has potential targets.

1.Introduction

Osteonecrosis of the femoral head (ONFH), also known as ischemic necrosis of the femoral head, is a chronic and multifactorial disease with a high disability rate and poor prognosis.It belongs to the categories of traditional Chinese medicine such as “bone obstruction”, “bone erosion”, and “bone flaccidity”.Epidemiological survey shows that the incidence rate of men is higher than that of women, and the incidence rate of overweight people or people who have been engaged in physical labor for a long time is higher[1], which tends to occur in young and middleaged people aged 20-40[2].Long term strain, external trauma, longterm use of hormones, long-term alcohol consumption, long-term exposure to radiation, overweight, and other factors are the main causes of avascular necrosis of the femoral head.If not treated in a timely manner, it can cause joint dysfunction, thereby reducing the quality of life of patients and reducing their life expectancy.For many years, the mechanism of femoral head necrosis has been continuously explored.Traditional Chinese medicine believes that“when evil energy accumulates, the qi mechanism is insufficient.”This indicates that various causes of the body can lead to imbalances in the organs, blood, and body fluids, leading to a decrease in the body’s resistance.After necrosis, the cartilage under the femoral head will be damaged, causing poor blood circulation and blocked meridians.[3]Modern medical theory categorizes this disease into three categories based on its etiology[4]: ① traumatic necrosis of the femoral head caused by fractures; ② Congenital dysplasia; ③Non traumatic femoral head necrosis caused by long-term excessive use of hormones, anemia, excessive alcohol consumption, etc.This disease is more common in the affected hip joint pain, with pain in the groin area being the most common, often found in the small arteries of the femoral head.In the later stages, due to excessive fatigue, the pain worsens, and even symptoms of claudication may occur.Currently, there is a large amount of scientific evidence indicating that traditional Chinese medicine plays an important role in studying the protective mechanism of femoral head necrosis,which can delay the progression of the disease, especially for patients in the early and middle stages.Therefore, this study aims to construct a model of traumatic femoral head necrosis in beagles,and conduct preliminary research using all Eucommia capsules to explore the roles of VEGF, bFGF, as well as bcl-2, bax, caspase-3,etc.in ischemic necrosis of the femoral head, providing a new approach for clinical prevention and treatment of femoral head necrosis, and highlighting the unique insights of traditional Chinese medicine in treating this disease.

2.Materials and Methods

2.1 Experimental animals

In this experiment, a total of 12 SPF grade male beagles were selected, aged between 16 and 24 months, and weighing between 9.6 and 12.3 kg.Experimental animal license number SCXK(Liao) 2014-0003.All experimental animals were raised separately at 3.5 m² The kennel is disinfected and fed on time every day,allowing for free movement.This experimental plan has been reviewed by the Animal Experiment Ethics Committee of Liaoning University of Traditional Chinese Medicine Affiliated Hospital (No.21000092019051).

2.2 Experimental drugs

Model drug: Tiletamine hydrochloride zolazepam hydrochloride 50 (France VirBac Group, batch number: 83887902); Sterilization water for injection (China Otsuka Pharmaceutical Co., Ltd.,National Pharmaceutical Approval Number: H12020025);Liquid nitrogen: Penicillin (North China Pharmaceutical Co.,Ltd., National Pharmaceutical Approval Number: H20033934);Gentamicin Sulfate Injection (Chenxin Pharmaceutical Co.,Ltd., National Pharmaceutical Approval Number: H37021973);Atropine for injection (Tianjin Jinyao Pharmaceutical Co., Ltd.,National Pharmaceutical Approval No.H12020382); Ketamine injection (Zhejiang Jiuxu Pharmaceutical Co., Ltd., National Drug Approval No.H20023609); Ceftriaxone Sodium (Shanghai Roche Pharmaceutical Co., Ltd., National Drug Approval No.H10983037).Experimental medication: Quan Du Zhong Capsules are provided by the Pharmacy Department of Liaoning University of Traditional Chinese Medicine Affiliated Hospital, with each capsule containing 0.48 g (equivalent to 2.5 g of the original medicinal material).Xianlinggubao Capsules are provided by the Pharmacy Department of Liaoning University of Traditional Chinese Medicine Affiliated Hospital, with a specification of 0.5g/capsule.

2.3 Experimental reagents and instruments

2.3.1 Experimental reagents

Experimental reagent: Hematoxylin eosin (HE) staining kit(Biyuntian Biotechnology, batch number: C0105); Anhydrous ethanol (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 100092008); Xylene (China National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number:10023418); Isopropanol (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 40049962); Ammonium persulfate(Beijing Suo Cai Bao Technology Co., Ltd., batch number A8090);Canine Vascular Endothelial Growth Factor A (VEGFA) ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., batch number: ml024667); Canine basic fibroblast growth factor (b-FGF)ELISA kit (Shanghai Enzymes Biotechnology Co., Ltd., batch number: ml061095); High sensitivity chemiluminescence detection kit (Kangwei Century Biotechnology Co., Ltd., batch number:CW0049); BCA Protein Assay Kit (Kangwei Century Biotechnology Co., Ltd., batch number: CW0014); PMSF (Beijing Suo Cai Bao Technology Co., Ltd., batch number: P0100); Lichun Red Staining Reagent (Kangwei Century Biotechnology Co., Ltd., batch number: CW0057S); RIPA protein lysis solution (strong) (Kangwei Century Biotechnology Co., Ltd., batch number: CW2333); two ×SuperStay sampling buffer (Kangwei Century Biotechnology Co.,Ltd., batch number: CW2636); Rabbit Antibody (Beijing Boorsen Biotechnology Co., Ltd.); TRIzon Reagent (Kangwei Century Biotechnology Co., Ltd., batch number: CW0580); ANti Å Bcl2L2(Boorsen, batch number: bs Å 5903R); ANti Å Bax (Beauson, batch number: hs-28034R); ANti Caspase 3 (Beauson, batch number bs 0081R).

2.3.2 Experimental instruments

SHZ-88 water bath constant temperature oscillator (from Lindbergh Instrument Manufacturing Co., Ltd.); SpectraMaxi3 multifunctional full wavelength enzyme-linked immunosorbent assay (manufactured by MD company in the United States), ST16R frozen high-speed centrifuge (manufactured by Thermo company); TAB-120FR fully automatic biochemical analyzer (manufactured by Toshiba Corporation in Japan); Leica (brand) BOND-MAX (model) fully automatic immunohistochemical staining agent; ASP 6025 fully automatic dehydrator (manufactured by Leica), RM2235 manual paraffin slicer (manufactured by Leica), EG1150H embedding machine (manufactured by Leica), HI1220 drying machine(manufactured by Leica), fully automatic biochemical analyzer(Hitachi 7180), M8 digital scanning microscopy imaging system(Precision, Germany); Vertical plate electrophoresis device (Bio Rad, USA); Trans Lot SD Semi Dry Transfer Printing (Bio Rad,USA), Chemiluminescence Imaging System (Shanghai Tianneng Technology Co., Ltd./5200)

2.4 Experimental Methods

2.4.1 Grouping and Modeling

Using the random number table method, 12 beagles were divided into 4 groups, namely 3 normal groups, 3 model groups, 3 whole Eucommia ulmoides capsules groups, and 3 Xianlinggubao capsules groups.Using a lateral lying position, make the midpoint of the line connecting the posterior superior iliac spine and the greater trochanter an arc as the cutting point.Cut the skin, subcutaneous tissue, and fascia in sequence, and then cut the fascia to fully expose it at the distal end.Separate along the direction of the muscle fibers to fully expose the external rotator muscle group, rotate the outer side of the femur, cut a small segment of the tendon of the external rotator muscle, place a sterile funnel cover on the femoral joint capsule, protect the surrounding soft tissue with sterile gauze,inject nitrogen every time until the liquid nitrogen is completely evaporated, and then warm it with physiological saline.

2.4.2 Surgical methods and administration

Routine alcohol disinfection is performed in the surgical area,with a sterile cavity towel placed around one side of the greater trochanter as the center, and an arc-shaped incision is made at the anterior edge of the femoral shaft; Cut the skin, expose the Femoral triangle area, separate the fat connected with the articular capsule,expose the articular capsule, expose the greater trochanter of the femur downward along the neck of femur, position and inject the needle.Use an orthopedic drill to insert a Kirschner wire about two centimeters below the femoral trochanter, and drill along the direction of the neck of femur into the femoral head for focus removal.Rinse after operation, then suture layer by layer, cover the operation area with sterile dressing, re fix with bandage, and send it back, and inject cefmetazole sodium intramuscularly once a day to prevent local infection.During the modeling period, there was no intervention in the normal group and model group.Convert according to the equivalent dose ratio table for converting the surface area of humans and animals in the “Methodology of Traditional Chinese Medicine Pharmacology Research (3rd edition)”.Administer the whole Eucommia capsule group once a day, with 2 whole Eucommia capsules taken once a day; The Xianlinggubao capsule group received two Xianlinggubao capsules once a day for 12 consecutive weeks.Start gastric lavage on the first day of modeling.

2.5 Observation indicators and methods

2.5.1 HE staining of canine femoral head tissue

Fix the canine femoral head specimen with 4% paraformaldehyde for more than 24 h and follow the conventional method of hematoxylin eosin (HE) staining.75%, 85%, 95%, and 100%gradients were used for alcohol dehydration, xylene transparency,paraffin immersion embedding, tissue block sectioning, xylene dewaxing, 100%, 95%, 85%, 75% gradient alcohol rehydration,hematoxylin staining, 70% hydrochloric acid alcohol differentiation,eosin staining, neutral gum sealing, and tissue morphology observation under a light microscope.

2.5.2 Detection of serum VEGF and BFGF

After 12 weeks of modeling in each group, blood was collected from the ear vein of beagle dogs using ELISA method and following standard procedures.

2.5.3 Micro CT detection

Apply Micro CT to scan and film the femoral head, and use the built-in software to calculate bone density (BMD), bone volume fraction (BV/TV), area per unit volume of bone tissue (BS/BV),trabecular thickness (Tb.Th), number of trabeculae (Tb.N), and trabecular space (Tb.SP).

2.5.4 Immunohistochemical detection of the expression levels of bcl-2, bax, and caspase-3

Take a paraffin section of the pathological femoral head of the dog(first fixed with 4% paraformaldehyde/0.1 mol PBS (pH 7.0～7.6) for 60 minutes, rinsed with PBS for 2 min, rinsed twice, and dehydrated at 5 ℃.Embed the sample with paraffin and slice, with a thickness of 8 μ m) Complete the mounting with glass slides and bake in a baking oven at 75 ℃ for 1 h.Place the first antibody reagent container to be prepared in the reagent rack, and then place the second antibody combination reagent rack and the first antibody combination reagent rack on the operating platform.

Use image J software to analyze the average optical density(AOD) values of each group, use the formula AOD=IOD/Area for quantitative comparative analysis of proteins, and use the average optical density value (AOD) to represent the relative content of different protein expressions in each group.

2.6 Statistical methods

Using SPSS 22 0 software for statistical analysis.The data is represented by (±s), and one-way ANOVA is used between groups.P＜0.05 indicates statistical significance.

3.Results

3.1 histopathology observation of femoral head in each group at the 12th week of modeling

The HE staining results showed that the calcified tissue of the femoral head in the normal group (A) was tight, and the bone trabeculae were arranged neatly.The proportion of calcified tissue in the femoral head of model group (B) is low, and the trabeculae are loosely arranged and irregular.The whole Eucommia ulmoides group (C) and Xianling Gubao capsule group (D) have multiple and tightly arranged trabecular bone tissues in dogs.And the trabeculae of the femoral head in the whole Eucommia ulmoides group are more closely arranged than those in the Xianling Gubao group.See Figure 1.

3.2 Levels of vascular endothelial growth factor (VEGF) in each group at week 12 of modeling

Quan Du Zhong Capsule can significantly upregulate the vascular endothelial growth factor of dog femoral head; Compared with the normal group, the serum VEGF level in the model group decreased after 12 weeks of modeling (P＜0.05); The normal group showed higher expression than the model group, but there was no significant advantage; Compared with the model group, the serum VEGF levels of the Xianling Gubao capsule group and the Quan Du Zhong capsule group increased at the 12th week of modeling (P＜0.05), and the difference between the two treatment groups was statistically significant (P＜0.05).The Quan Du Zhong capsule group had the highest level, followed by the Xianling Gubao capsule group.See Table 1.

Fig 1 HE staining results of femoral head tissue in dogs of each group(× 100)

Tab 1 Expression level of VEGF-A in each group(pg/mL,±s)

Tab 1 Expression level of VEGF-A in each group(pg/mL,±s)

Compared with the normal group, *P＜0.05, **P＜0.01; Compared with the model group, △P＜0.05, △△P＜0.01

Group VEGF-A content(pg/mL)normal group 477.50±33.06 model group 359.80±31.16**QuanDuZhong Capsule group 525.40±35.82△△Xianlinggubao Capsule group 509.80±64.35△△

3.3 Level of basic fibroblast growth factor (BFGF) in each group at week 12 of modeling

Quan Du Zhong capsule can significantly upregulate the basic fibroblast growth factor of dog femoral head; Compared with the normal group, the serum BFGF level in the model group decreased after 12 weeks of modeling (P＜0.05); The normal group showed higher expression than the model group, but there was no significant advantage; Compared with the model group, the Xianling Gubao Capsules group and the Quan Du Zhong Capsules group showed an average increase in serum BFGF water at the 12th week of modeling(P＜0.05), and the difference between the two treatment groups was statistically significant (P＜0.05).The Quan Du Zhong Capsules group had the highest level, followed by the Xianling Gubao Capsules group.See Table 2.

Tab 2 Expression levels of BFGF-A in each group(pg/mL,±s)

Tab 2 Expression levels of BFGF-A in each group(pg/mL,±s)

Compared with the normal group, *P＜0.05, **P＜0.01; Compared with the model group, △P＜0.05, △△P＜0.01

Group BFGF content(pg/mL)normal group 73.17±6.013 model group 51.58±3.343**QuanDuZhong Capsule group 96.67±5.399△Xianlinggubao Capsule group 92.92±5.518△△

3.4 Comparison of parameters and indicators of Micro CT scanning of femoral head in each group at week 12 of modeling

From Table 3, it can be concluded that compared with the normal group, the BMD, BS/BV, BV/TV, Tb.Th, and Tb.N of the model group will be lower, while Tb.SP will be higher, with statistical significance (P＜0.05).Compared with the model group, the Xianling Gu Bao capsule group and the Quan Du Zhong capsule group showed an increase in BMD, BV/TV, Tb.N, and Tb.Th, while Tb.SP and BS/BV decreased, with statistical significance (P＜0.05).

Tab 3 Comparative parameter indicators of Micro CT scanning of femoral head in each group at week 12 of modeling(±s)

Tab 3 Comparative parameter indicators of Micro CT scanning of femoral head in each group at week 12 of modeling(±s)

Parameter indicators normal group Xianlinggubao Capsule group QuanDuZhong Capsule group model group BMD(mg/cm^3) 1 042.36±47.28 958.357±83.28 943±57.67 819.37±49.23 BS/BV(1/mm) 18.67±2.59 11.63±1.87 11.32±2.41 13.68±1.83 BV/TV(%) 75.32±5.68 68.29±5.81 67.39±5.95 48.927±4.49 Tb.Th(mm) 0.48±0.04 0.37±0.05 0.34±0.04 0.26±0.03 Tb.Sp(mm) 0.12±0.03 0.16±0.03 0.19±0.04 0.298±0.06 Tb.N(1/mm) 1.71±0.06 1.67±0.08 1.49±0.17 1.24±0.2

3.5 Comparison of relative expression levels of bcl-2 protein in each group at week 12 of modeling

See Figure 3 and Table 4

The Quan Du Zhong capsule group and Xian Ling Gu Bao capsule group significantly increased the expression of bcl-2 protein in the femoral head of dogs, with a statistically significant difference compared to the model group (P＜0.05)

Tab 4 Bcl-2 expression level(±s)

Tab 4 Bcl-2 expression level(±s)

Group BCL-2 content(pg/ml)normal group 1.426±0.1384 model group 0.5619±0.06485**QuanDuZhong Capsule group 0.8038±0.09519△Xianlinggubao Capsule group 0.8271±0.1302△△

3.6 Comparison of relative expression levels of bax protein in each group at week 12 of modeling

See Figure 4 and Table 5

The Quan Du Zhong capsule group and Xian Ling Gu Bao capsule group can significantly reduce the expression of bax protein in the femoral head of dogs, which is statistically significant compared to the model group (P＜0.05)

Tab 5 Bax expression level(±s)

Tab 5 Bax expression level(±s)

Group Bax content(pg/mL)normal group 0.737±0.1433 model group 1.767±0.1884**QuanDuZhong Capsule group 1.127±0.121△Xianlinggubao Capsule group 1.051±0.1186△△

Fig 2 Immunohistochemical map of Bcl-2 and expression of average optical density of Bcl-2 in each group

3.7 Comparison of relative expression levels of caspase-3 in each group at week 12 of modeling

See Figure 5 and Table 6

The Quan Du Zhong capsule group and Xian Ling Gu Bao capsule group can significantly reduce the expression of caspase-3 protein in the femoral head of dogs, which is statistically significant compared to the model group (P＜0.05)

Tab 6 Caspase-3 expression level

Fig 4 Immunohistochemical map of caspase-3 and expression average optical density of caspase-3 in each group

4.Discussion

Currently, ONFH is the most common type of hip joint disease.Multiple reasons can cause ischemia of the femoral head, leading to necrosis, collapse, and other pathological changes.If not treated in a timely manner, it can develop into osteoarthritis in the later stage.The disease has a high incidence rate.Today, the disease has increasingly become a global health problem, and we currently have no perfect treatment.When the disease progresses to the late stage,hip replacement surgery is usually performed.Although the surgical effect is significant, the lifespan of the implant is limited, and it may require revision and the cost of surgery is also high[5].Therefore,such patients often hesitate in the late stage of the disease.On the other hand, some patients are unable to undergo surgery due to their underlying diseases.In addition, the secondary trauma caused by surgery is inevitable.Therefore, currently, hip replacement surgery is not a good treatment option for adolescent patients with femoral head necrosis.Therefore, finding a better prevention and treatment method for patients with femoral head necrosis and reversing early femoral head necrosis has become a trend.

Xianlinggubao capsules are often used for orthopedic and traumatology diseases.They contain effective ingredients such as Epimedium, Salvia miltiorrhiza, Dipsacus, Psoralea, Rehmannia glutinosa, and Anemarrhena asphodeloides.They have the effects of tonifying the liver and kidney, strengthening tendons and bones,and promoting blood circulation and unblocking collaterals.Zhao Dingyan et al[6] speculated through experimental results that icariin can intervene in the progression of femoral head necrosis.Jia Bingshen et al[7] found through experiments on rats that icariin has a significant improvement effect on femoral head necrosis and can repair necrotic tissue.And studies[8-9] have shown that Xianling Gubao Capsule can improve blood circulation and function of the hip joint, reduce patient pain, improve bone metabolism levels,and is widely used in clinical practice.Therefore, this experiment uses Xianling Gubao Capsule as a positive comparison to explore the therapeutic effect of Quan Du Zhong Capsule on femoral head necrosis.

In the “Shennong Materia Medica Classic”, Eucommia ulmoides is referred to as the top grade in medicine.Eucommia ulmoides has high clinical value and is widely used.At present, traditional Chinese medicine treatment for femoral head necrosis is very popular and has higher safety.Among all Eucommia capsules, all Eucommia ulmoides has high medical value as its active ingredient.Eucommia ulmoides gum forms an excellent “bone adhesive”due to its high tensile strength.The “Shennong Materia Medica Classic” once recorded that Eucommia ulmoides has a warm nature and has functions such as strengthening tendons and bones,tonifying liver and kidney, tonifying waist and knees, and dispelling soreness[10].Its chemical composition mainly includes lignans,iridoids, phenylpropanoids, flavonoids, polysaccharides, Eucommia ulmoides gum, and antifungal proteins[11], which can effectively treat bone diseases such as fractures and osteoporosis.Liu Dong observed several patients with knee osteoarthritis and found that Quan Du Zhong Capsule can effectively improve knee joint function and alleviate pain[12].And Zhang Wenbo et al.believed that M2 macrophage supernatant can be used as an auxiliary tool for tissue material engineering by studying its binding to total flavonoids of Eucommia ulmoides[13].

The establishment of animal model of femoral head necrosis is the basis of a successful experiment.At present, there are many experimental animals and modeling methods.The physiological characteristics, tissue and anatomical structure of dogs are similar to that of humans, with high comparability.In addition, the canine femoral head necrosis model prepared by liquid nitrogen freezing method is widely used.The use of liquid nitrogen low temperature causes vasospasm around the femoral head, destroys endothelial cells, and then leads to femoral head necrosis[14].Therefore, in this experiment, 12 beagles were selected as model animals to establish an ONFH model through liquid nitrogen perfusion,and full Eucommia ulmoides capsules were administered for intervention.Through HE staining, the results showed that at week 12 of modeling, HE staining showed that the calcified tissue of the femoral head in the normal group of dogs was tight, and the bone trabeculae were arranged neatly.The proportion of calcified tissue in the femoral head of the model group dogs is low, and the bone trabeculae are loosely arranged and irregular[15].The above results indicate that we have successfully established the ONFH model.

Apoptosis is the active death of cells in order to maintain balance in the body.In ischemic necrosis of the femoral head, osteoblasts play a crucial role in the formation, metabolism, and mineralization of bone tissue.When bones are damaged, osteoblasts quickly generate new bone tissue to repair bone damage.If the osteogenic necrosis are necrotic, this process will be blocked, which will lead to necrosis of the femoral head, thus affecting its function.There is a complex apoptosis balance between osteoblasts and osteoclast.If the imbalance affects a series of reactions, it is extremely important to maintain the dynamic balance between them.Therefore,controlling osteoblast death is a crucial step in inhibiting femoral head necrosis.Apoptosis is regulated by multiple genes.Currently,bcl-2 and bax are a pair of genes that regulate apoptosis.bcl-2 can inhibit apoptosis, while bax can inhibit the effect of bcl-2.Bcl-2 binds to bax to form homologous dimers[16].Most studies have found that the ratio between these two proteins is an important factor in determining cell apoptosis to a certain extent.Under the stimulation of apoptosis signals, bax is activated to form a dimer on mitochondria, which enhances mitochondrial membrane permeability and leads to cell apoptosis.Caspase-3 also plays a crucial role in the process of cell apoptosis.Research has shown that reducing the activation of caspase-3 can effectively inhibit the progression of cell apoptosis.We demonstrated the interaction between bcl-2, bax, and caspase-3 by detecting the expression of all Eucommia capsules, which significantly reduced the expression of caspase-3 protein and inhibited the apoptosis process.From the experimental results, we can infer that Quanduzhong capsule has the following effects: inhibit the activity of osteoclast, enhance the survival of osteocytes, and thus accelerate the proliferation of bone.

Necrosis of the femoral head is caused by a decrease in blood supply, resulting in hypoxia and necrosis.To delay the process of femoral head necrosis, it can be achieved by releasing vascular endothelial growth factor, leading to angiogenesis and osteogenesis[17], which is an effective way.VEGF is an important factor in this process.VEGF, originally known as vascular permeability factor,was isolated from tumor cells by Senger et al.in 1983[18].VEGF has the effect of promoting endothelial growth, enhancing permeability,and proliferation, and is the strongest angiogenic factor.The relationship between bone and blood vessels is very close, and blood vessel development occurs earlier than bone[19].The experimental results of Yang Cao et al.[20] indicate that gene transfection can promote vascular regeneration in bone tissue, thereby further improving bone regeneration.At the same time, vascular endothelial growth factor can promote Vascular permeability, which can be used as a new method to promote fracture repair[21].Bi Kai et al.[22]demonstrated through experiments on rabbits that the expression of VEGF and bFGF in different tissues can promote the expression of VEGF during fracture treatment, and staged treatment of fractures.Further clinical applications will provide a feasible and non-invasive treatment method for fractures.And research has shown that VEGF can significantly increase the area and quantity of microcirculation in bone, thereby increasing the blood supply in bone and providing essential nutrients for bone metabolism[23].BFGF also plays an important role in bone cell proliferation and local blood reconstruction.These growth factors can promote the regeneration of new blood vessels and promote the repair of necrotic sites.The pharmacological research results of Xianlinggubao Capsule show that it can promote bone formation[24], accelerate the remodeling speed of bone callus mineralization, upregulate the level of vascular endothelial growth factor to repair the microvessels of the femoral head, thereby protecting bone cells and inhibiting the occurrence of femoral head necrosis.We detected the content of vascular endothelial growth factor in the femoral head of dogs using ELISA method, and it was evident that VEGF and bFGF were lower in the necrotic femoral head of dogs in the model group than in the whole Eucommia ulmoides group and Xianling Gubao group.So based on the experimental results, we can infer that Quan Du Zhong Capsule and Xian Ling Gu Bao Capsule have similar effects, which can effectively resist bone necrosis, improve local blood circulation of the femoral head, and treat femoral head necrosis.

In summary, Quan Du Zhong Capsule can increase the expression levels of VEGF and bFGF in serum, increase the amount of bcl-2 protein, inhibit the amount of bax protein, and reduce the amount of caspase-3 protein, reduce the occurrence of cell apoptosis, stabilize the internal environment, and delay the progression of avascular necrosis of the femoral head.It has a synergistic effect on the treatment of avascular necrosis of the femoral head and has potential therapeutic targets.On this basis, we will further combine clinical treatment effects to provide new ideas for more effective prevention and treatment of ONFH.