LI Sheng, ZHU Cai-yu, GU Yi-fan, ZHU Lei, LI Zi-peng, CHEN Shao-qi, ZHOU Zheng-xin?

1.First Clinical Medical College of Anhui University of Traditional Chinese Medicine, Hefei 230031 ,China

2.The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China

ABSTRACT Objective: To investigate the therapeutic effect of osteopathic osteopathy on hormonal osteonecrosis in rabbits by regulating the Wnt/PI3K-AKT signaling pathway.Methods: Thirty clean-grade New Zealand rabbits were randomly divided into control group, model group and experimental group, and all except for the control group, allogeneic serum combined with glucocorticoids were used to establish animal models of simulated hormonal osteonecrosis of the femoral head.After successful modeling, the experimental group was given the model group and the control group the same amount of normal saline, and the experimental group was given bone palsy granule concentrate according to the equivalent dose of humans and rabbits, once a day for 8 weeks, and after intraperitoneal anesthesia, blood was collected from the extracorporeal heart and the serum ALP and TGF-β1 levels were detected by immunoenzyme-linked adsorption.The experimental animals were sacrificed,，and the bilateral femoral heads of rabbits were removed for hematoxylin-eosin staining, and the apoptosis of cells in the femoral head area was observed by TUNEL staining.RT-qPCR analyzed the expression of Bax and Bcl-2 genes, and Western blot detected the expression levels of pathway proteins Wnt, β-catenin, GSK-3β, AKT and p-AKT.Results: Compared with the control group, the expression levels of ALP and TGF-β1 in rabbit bone tissue in the model group decreased significantly (P＜0.05), the bone trabeculae were sparse and thinned,the number of hollow bone fosses and the number of apoptosis-positive cells increased significantly (P＜0.05), the expression of pro-apoptotic gene Bax was increased, and the expression of anti-apoptotic gene Bcl-2 was inhibited (P＜0.05).After the intervention and treatment of bone palsy granules，the hollow bone depression and apoptosis of rabbit bone tissue in the experimental group were effectively improved (P＜0.05).In addition, compared with the model group, the osteopathic elimination particles could upregulate the expression of ALP and TGF-β1 (P＜0.05), promote the expression of pathway proteins Wnt and β-catenin, increase the phosphorylation level of AKT, and downregulate the expression of GSK-3β (P＜0.05).Conclusion: Under glucocorticoids, osteoblast development dysfunction and apoptosis increase.Bone palsy granules can regulate the two signaling pathways of Wnt/PI3K-AKT, promote osteoblast development and inhibit apoptosis, effectively maintain bone metabolic balance, prolong the collapse time of the femoral head, and further treat osteonecrosis of the femoral head.

Keywords:

Osteonecrosis of the femoral head

Bone palsy eliminates granules

Glucocorticoids

Osteoblasts

GSK-3β

1.Introduction

Necrosis of the femoral head is a clinically refractory bone and joint disease, which results in blood flow disorder of the femoral head and ischemic necrosis of bone cells and bone marrow components due to various reasons[1].Necrosis of the femoral head often occurs in young and middle-aged people aged 30-50 years.The primary reason for patients to see a doctor is often pain in the hip joint.Under the effect of continuous stress, there will also be dysfunction.Once the disease worsens, it will even cause irreversible joint disability[2].In recent years, glucocorticoids have been more and more widely used in anti-inflammatory and immune regulation, resulting in steroid-induced avascular necrosis of the femoral head (SANFH) accounting for an increasing proportion of non-traumatic necrosis of the femoral head.In China alone, the number of patients with non-traumatic necrosis of the femoral head is up to 8.12 million[3].Therefore, if the role of pathogenic factors can be eliminated or blocked in time at the early stage of steroidinduced femoral head necrosis, it will be the most effective and active prevention and treatment measures.

Wnt / β- Catenin pathway, also known as the classic Wnt signaling pathway, plays a key role in the treatment of SANFH by regulating the occurrence and development of SANFH, stimulating the development of bone cells and inhibiting apoptosis[4].In recent years, more and more studies have shown that PI3K/AKT pathway can promote angiogenesis and protect osteoblasts from GCs-induced apoptosis[5].On this basis, our research team found that Gubi Tongxiao granule can regulate Wnt/ β- Catenin pathway promotes the increase of bone mass by promoting the renewal of bone marrow mesenchymal stem cells (BMSCs), inducing the formation of osteoblasts, and inhibiting the apoptosis of osteoblasts.It is also found that Gubitongxiao granules can interfere with the PI3K-AKT signal pathway, promote the proliferation of osteoblasts, and play a regulatory role in promoting cell proliferation and differentiation[6-7].GSK-3 β It is the common downstream substrate of the two signal pathways, and plays a very important role in the transduction of their respective pathways.Therefore, the research team proposed that Gubitongxiao granules may participate in the regulation of these two pathways in the treatment of steroid-induced femoral head necrosis at a certain dose.Therefore, this experiment, through the preparation of SANFH rabbit model, detected the expression of the related proteins of the two pathways, To explore the possible mechanism of Gubitongxiao granule in interfering with SANFH at the cellular and molecular level.

2.Materials and methods

2.1 Experimental materials

2.1.1 The laboratory animals

Were purchased from Anhui Experimental Animal Center(production license No.SYXK (Anhui) 2020-001), 30 healthy 3-month-old, adult male detergent New Zealand white rabbits,weighing (2.5-3.0kg), and the animal certificate number was NO.202213666.According to the requirements of animal ethics,feed with common feed and suspend drinking water (ethics number:AHUCM-rabbits-2022020).

2.1.2 The experimental drug

Gubitongxiao Granules (Dipsacus asperus 10 g, Epimedium epimedium 10 g, Cinnamomum cinnamomi 4 g, Angelica sinensis 10 g, Ligusticum chuanxiong 10 g, Salvia miltiorrhiza 15 g, Paeonia rubra 15 g, ground beetle beetle 6 g, Glycyrrhiza uralensis 6 g) were produced and purchased by the Traditional Chinese Medicine Store of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, and dexamethasone sodium phosphate for injection (Chongqing Laimei Pharmaceutical, 5mg/tube) were purchased from the Western Pharmacy of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.

2.1.3 Experimental reagent and instrument

Horse serum (Biyuntian Biology, product No.: C0262); ALP、TGF- β 1 Enzyme-linked immunosorbent kit (enzyme-linked organisms, product number: ml095274, ml028093); TUNEL Apoptosis Detection Kit (Xavier Biology, Product No.: G1504-50T);Cell RNA rapid extraction kit (Shandong Sikejie Biotechnology Co., Ltd.Product No.: AC0205-A); Reverse transcriptional and real-time fluorescence quantitative PCR kit (New shellfish, product No.: R202, Q204), tissue lysate (Biyuntian Biological, product No.:S3090); SDS-PAGE gel rapid preparation kit (Shandong Sikejie Biotechnology Co., Ltd.Product No.: P1200 EC0004); Wnt、βcatenin、GSK-3 β、AKT and p-AKT antibodies were purchased from Bios (product number: bs-6148R, bs-1165R, bs-0028R, bs-0115R, bs-0876R); Full-band enzyme marker (BioTek, USA,model: Epoch 2); Tissue dehydrator, embedding machine, paraffin sectioner (Zhejiang Jinhua Huiyou Instrument and Equipment Co., Ltd., model: HY-TS1090B, HY-BM1160, HY-3500); Inverted fluorescence microscope (Guangzhou Minmei Photoelectric Technology Co., Ltd., model: MF53-N); Real-time fluorescence quantitative PCR instrument (Suzhou Yarui Biotechnology Co., Ltd.model: MA-6000); WB gel imaging analysis system (Bio rad, USA,model: ChemiDoc).

2.2 Experimental method

2.2.1 Animal model establishment and intervention

30 New Zealand white rabbits were fed adaptively for one week,and then randomly divided into control group, model group and experimental group, with 10 rabbits in each group.In addition to the control group, each rabbit was injected with horse serum through the ear vein once a week.After 4 weeks, dexamethasone was injected intramuscularly.The left and right gluteal muscles were injected alternately four times, once every 24 h.During the period, penicillin 800 000 U and gentamicin 80 000 U were injected intramuscularly to prevent infection.After confirming the success of modeling, prepare Gubi Tongxiao Granule Concentrate (granules provided by the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine), according to the equivalent dose of rabbits=[(human drug measurement) 119 g/70 kg × 3.27=(rabbit drug measurement) 5.56 g/kg], fed twice a day.After 8 weeks of feeding, blood was collected from the heart in vitro after abdominal anesthesia and serum was aseptically separated, and stored in a refrigerator at -20 ℃ for standby.After the experiment, the experimental animals were killed according to the regulations, and the femoral heads of both rabbits were taken out, soaked in 4% paraformaldehyde, and stored in a constant temperature refrigerator at -20 ℃ for future use.The animal carcasses were sent to the Experimental Animal Center of Anhui University of Traditional Chinese Medicine for unified treatment.

2.2.2 Detection of ALP and TGF in rabbit serum by ELISAβ1 Activity

Take out the Flat noodles that have been balanced at room temperature for 60 min, set up the test sample hole, model sample hole and control sample hole, add 50 μL of sample diluent, standard sample and sample to be tested respectively, add 100 μL of detection antibody to each hole, seal the hole, incubate and wash the plate,add substrate to each hole and incubate for 15 min, add 50 μL of termination solution to each hole, and measure the OD value of each hole at the wavelength of 450 nm within 15 min.

2.2.3 Observe the histopathological conditions by HE staining

Decalcify the paraffin section with 10% formic acid solution.After completion, embed, slice and bake the paraffin section in turn.After dewaxing and hydration, conduct hematoxylin-eosin staining.After completion, conduct gradient dehydration on the section, and use xylene to transparently treat the tissue section for 3 times, and then seal the section.Observe the specimen with a common microscope and take and save the image, Finally, observe the internal structure and empty bone lacuna in each tissue section under the optical microscope and calculate the empty bone lacuna rate (empty bone lacuna rate=the sum of all empty bone lacunae in 10 fields/the sum of all empty bone lacunae)

2.2.4 TUNEL staining method was used to observe the apoptosis of the cells in the femoral head area

The femoral head tissue samples were made into paraffin sections.After the dewaxing treatment was completed at room temperature,the tissue sections were placed in 20 μg/mL protease K solution, and after PBS rinsing, TUNEL mixed solution was added to fully react;Incubate in a 37 ℃ incubator for 2 h, remove the reaction solution,and then soak and wet the slices with PBS twice; After the residual protease K was washed with 0.1% Triton X-100 buffer (containing 5 mg/ml BSA), the apoptosis of the femoral head tissue in the section field was observed under the fluorescence microscope.

2.2.5 RT-qPCR analysis of osteogenesis related gene expression

In femoral head tissue and selection of related genes Bax and Bcl-2 as detection targets, β- Action is an internal parameter.Total intracellular RNA was isolated from tissues by using Trizol reagent.The total RNA of cells was reverse transcribed with cDNA synthesis kit, and then PCR amplification was performed with SYBR-GreenqPCR-Mix.After the reaction, the Ct value of each tested gene is derived, and the data analysis uses 2-ΔΔCtrelative expression method.

Tab 1 Primer sequence of each gene

2.2.6 Western blot is used to detect the expression of related proteins

And select related proteins Wntβ- catenin、GSK-3β、AKT and p-AKT are detection targets, β- Action is an internal parameter.According to the instructions of the kit, total protein was extracted from cells and tissues using RIPA solution.Carry out sample loading, gel electrophoresis, membrane rotation and sealing in turn,incubate the first antibody at 4 ℃ and prepare a shaker overnight,incubate the HRP labeled IgG antibody at room temperature for 2 h the next day, fully wash the membrane with TBST solution, place the PVDF membrane in the chemiluminescence system for detection,and use Image J software to conduct quantitative analysis of the strip.

2.2.7 Statistical analysis

All the experiments were repeated three times, and the data were statistically analyzed and described by using the statistical software SPASS25.0 (±s).The data were statistically analyzed by one-way ANOVA or rank sum test, and P＜0.05 was used to indicate that the difference was statistically significant.

3.Results

3.1 The general morphological observation of rabbits in each group

Showed a normal increase in the body mass of the three groups of animals with the extension of feeding time.After successful modeling, the growth trend of the control group was normal, while the body mass of the other groups decreased.As time went on,the spirit of the model group became more and more depressed,the activity decreased, the appetite decreased, the hair color was dim, the luster was poor, and the hair was easy to fall off.With the prolongation of medication time, the activity of the experimental group increased, appetite increased, body weight and hair color gradually recovered, but still slightly worse than the control group.

3.2 Serum ALP and TGF of rabbits in each group-β1

Compared with the control group, the ALP and TGF in the rabbit bone tissue of the model group- β The level of ALP and TGF in the experimental group decreased (P＜0.01)- β1 level increased, but there was still a certain difference compared with the control group(P＜0.05).

Tab 2 ALP and TGF in rabbit bone tissue of each group-β1 Expression level(n=3, ±s)

Tab 2 ALP and TGF in rabbit bone tissue of each group-β1 Expression level(n=3, ±s)

注：與對(duì)照組相比，**P＜0.01;與模型組對(duì)比，##P＜0.01.

組別 ALP(μg/L) TGF-β1(pg/mL)對(duì)照組 48.294±3.521 128.547±26.067模型組 26.238±4.425** 55.074±112.270**實(shí)驗(yàn)組 37.761±4.951## 91.639±12.933##F 19.389 12.179 P 0.002 0.008

3.3 Histopathological changes of rabbit bone in each group

After HE staining, it can be seen under the microscope that the shape of bone trabeculae in the control group is thick and orderly arranged; The arrangement of bone trabeculae in the model group was sparse, and the number of empty bone lacunae was significantly higher than that in the control group (P＜0.01).After the intervention of Gubitongxiao granule, the bone trabeculae in the experimental group were complete in shape and regular in arrangement, and the number of empty bone lacunae was significantly lower than that in the model group (P＜0.01).(See Figure 1 and Table 3).

Fig1 Histopathological observation of femoral head in each group (HE×50)

Tab 3 Comparison of hollow bone lacunae rate of rabbit femoral head tissues in each group(n=3,±s)

注：與對(duì)照組相比,**P＜0.05;與模型組對(duì)比, ## P＜0.05

組別 空骨陷窩率(%)對(duì)照組 14.958±1.849模型組 24.675±1.741**實(shí)驗(yàn)組 19.364±2.667##F 15.708 P 0.004

3.4 Apoptosis in the femoral head area of rabbits in each group

The nucleus of apoptotic cells was stained brown under TUNEL staining.Under the microscope, the number of brownish yellow nuclei in the model group was significantly higher than that in the control group (P＜0.01).(See Figure 2 and Table 4)

Tab 4 Comparison of apoptosis of rabbit femoral head area in each group (n=3,±s )

Tab 4 Comparison of apoptosis of rabbit femoral head area in each group (n=3,±s )

注：與對(duì)照組相比,**P＜0.01；與模型組對(duì)比,##P＜0.01

組別 凋亡率(%)對(duì)照組 3.841±0.192模型組 19.012±0.665**實(shí)驗(yàn)組 10.103±0.358 ##F 859.434 P P＜0.01

3.5 According to the results of RT-qPCR experiment

Bax mRNA expression in the model group was significantly higher than that in the control group, while Bcl-2 mRNA expression was inhibited (P＜0.01), resulting in changes in Bax/Bcl-2 ratio; After intervention treatment with Gubitongxiao granules, the above gene expression trend was reversed in the experimental group, but there was still a certain difference compared with the control group(P＜0.05) (see Figure 5 and Table 5)

3.6 Expression level of pathway related protein

Wnt β- The expression of Catenin, AKT and p-AKT protein were significantly lower than that of the control group, GSK-3 β Compared with the control group, Wnt β- The expression of Catenin, AKT and p-AKT in the model group was higher than that in the control group, but there were still deficiencies compared with the control group.GSK-3 β It was lower than the model group and higher than the control group (P＜0.05).(See Figure 4 and Figure 5)

Tab 5 mRNA expression level of femoral head tissue in each group (n=10,±s)

Tab 5 mRNA expression level of femoral head tissue in each group (n=10,±s)

注：與對(duì)照組相比,**P＜0.01；與模型組對(duì)比,##P＜0.01

組別 Bax Bcl-2對(duì)照組 1.009±0.066 1.002±0.022模型組 1.551±0.107** 0.587±0.064**實(shí)驗(yàn)組 1.194±0.061## 0.894±0.028##F 34.983 77.865 P＜0.01 ＜0.01

Fig 3 Mrna expression of VEGF, eNOs.Bcl-2 and Bax in each group

Fig 4 Western blot analysis of femoral head tissue protein in each group

Fig 5 Relative expression of proteins in each group after correction with internal reference protein

4.Discussion and outlook

At present, there is no very accurate view on the mechanism of osteonecrosis of the femoral head.There are mainly the following mainstream theories, such as osteoporosis, intravascular coagulation,lipid metabolism disorder, osteocyte apoptosis, and bone marrow mesenchymal stem cell differentiation abnormalities.Among them, GCs induce osteoblast apoptosis, leading to bone loss or even collapse of the femoral head is a topic of much discussion in recent years [8-9].In addition, in the context of SARS and COVID-19 epidemic, hormones are widely used in clinical practice, which makes the incidence rate of SANFH gradually increase and the age of onset tends to be younger, while early conservative treatment is crucial and essential for the middle and late irreversible hip joint disability[10-11].In the early conservative treatment of SANFH,traditional Chinese medicine has rich clinical experience and good curative effect[12].The disease can be classified as "bone erosion" in traditional Chinese medicine.The article of "Su Wen ·Bi Lun" said:"Wind, cold and dampness are mixed together, the disease is in the bone, bone marrow ache, and bone erosion"."Kidney deficiency"is the core of bone erosion, phlegm and dampness block is the key pathogenesis, blood stasis is the pathogenic factor, and is closely related to the kidney and spleen.Kidney deficiency is easy to cause blood stasis, spleen deficiency is easy to produce phlegm and dampness, qi and blood biochemical lack of source, limb joint loss of nourishment, pulse channel astringency is not smooth, blockage is pain, and dishonor is pain, Pain is the main clinical manifestation of ONFH[13].The traditional Chinese medicine Gubi Tongxiao Granules is based on Professor Ding E's experience.According to the fact that kidney deficiency is the root of bone erosion, the kidney tonic drugs such as Epimedium and Dipsacus are selected to replenish the body and cure the disease.At the same time, the sweet and moist angelica is used to combine yin and yang and seek yang in yin.In addition, salvia miltiorrhiza, ligusticum chuanxiong and red peony root have the function of promoting blood circulation and removing blood stasis.ground beetle beetle has the ability of breaking blood circulation and removing blood stasis.Four drugs are used together to smooth the blood vessels, dissipate blood stasis, and cinnamon is used to warm the body and remove cold,dredge the channels and relieve pain.Then, the old people can help to coordinate various drugs, tonify the heart and spleen, restrict the toxicity of ground beetle beetle, and finally form a formula[14].The previous study of the research group found that Gubitongxiao granule has an obvious regulatory effect on SANFH[15].This prescription plays a positive role in improving the tissue morphology of necrotic femoral head by inhibiting cell apoptosis, regulating bone metabolism, and maintaining bone mass stability.

The source of ALP is mainly secreted by osteoblasts and synthesized by the liver.It is an extracellular enzyme of osteoblasts and is closely related to the proliferation and differentiation of osteoblasts and the normal development of bone.After released by osteoblasts, ALP can hydrolyze inorganic phosphate, reduce the concentration of pyrophosphate, and promote bone mineralization.Therefore, when the content of ALP in serum is low, it indicates that bone mineralization is blocked[16].TGF- β It is synthesized and secreted by osteoblasts and chondrocytes, which can regulate the proliferation and differentiation of osteoblasts and play a significant role in promoting bone formation.At the same time, it has a certain inhibitory effect on osteoclasts and can promote the formation of chondrocytes.TGF in serum- β 1.Decreased, indicating osteoporosis [17-18].In this study, after successful animal modeling,ALP and TGF in rabbit bone tissue of model group- β 1 The content is the lowest, ALP and TGF in the experimental group- β The content of 1 was significantly increased (P＜0.05), which still had a certain gap compared with the control group.After HE staining,under the microscope, it can be seen that the bone trabeculae in the model group are sparse and thin, scattered, and there are many empty bone lacunae.The rate of empty bone lacunae in the model group is significantly higher than that in the control group (P＜0.01).However, after the intervention of Gubitongxiao granule, the staining section of rabbit femoral head tissue in the experimental group showed that the body of bone trabecula was thick, large in number,compact in arrangement, and the rate of empty bone lacuna was significantly lower than that in the control group (P＜0.01).

The study found that Bax and Bcl-2 are a pair of genes that control cell apoptosis.Bax is a pro-apoptotic member of the Bcl-2 gene family.It regulates the endogenous apoptosis of cells by encoding specific proteins, while Bcl-2 can prevent cell death by regulating cell apoptosis and prolong the survival time of aging cells.In the repair of femoral head necrosis, it can delay bone necrosis and alleviate bone loss[19-20].In this experiment, TUNEL staining showed that apoptotic nuclei were stained brown and distributed unevenly among normal cells in a scattered form.Microscopic observation showed that the number of apoptotic nuclei in the visual field of the model group was increased and the level of apoptosis was significantly higher than that of the control group (P＜0.01);However, the apoptosis of the rabbit model in the experimental group was significantly improved after the intervention of Gubitongxiao granules (P＜0.01).

Relevant research shows that Wnt/ β- Catenin pathway plays an important role in the regulation of SANFH, promoting glycogen synthesis kinase 3 by regulating the related receptors on the cell membrane surface β (GSK-3 β) Phosphorylation β- Catenin degrades, making a large amount of accumulated β- Catenin enters the nucleus to further activate downstream target genes, thus accelerating the proliferation and differentiation of osteoblasts[21-22].Therefore, in this pathway, GSK-3 β Activation and β-The content of Catenin is very important.GSK-3 β It is also the downstream kinase of PI3K/AKT pathway.When PI3K/AKT is activated, it will phosphorylate phosphoinositol protease and then express it downstream to activate GSK-3 β[23].On the one hand,it can enhance Wnt/ β- The ability of ctenin signal pathway to promote osteogenic differentiation; On the other hand, studies have shown that PI3K/AKT pathway can maintain bone balance by inhibiting osteoclast proliferation[24].Chen[25] and others found that panax notoginseng saponins can activate Wnt/ β- Catenin path lifting LRP5 and β- The expression of Catenin protein can promote the proliferation of osteoblasts and inhibit apoptosis, thus playing a role in bone protection.Song [26] and others found that PI3K/AKT can regulate GSK-3β Impact Wnt/ β- Catenin has the possibility of enhancing osteogenic differentiation.In this experiment, Gubi Tongxiao Granule significantly up-regulated Wnt β- Catenin, AKT,p-AKT and other proteins down-regulate GSK-3 β Protein, promote osteoblast differentiation and inhibit cell apoptosis.It is suggested that Gubitongxiao granule may activate Wnt at the same time/ β-Catenin and AKT/p-AKT signal pathway regulate SANFH.

To sum up, Gubi Tongxiao Granule may regulate cell apoptosis by simultaneously regulating the two signal pathways Wnt/PI3KAKT, alleviate the collapse time of the femoral head, delay bone necrosis and bone loss, promote the repair of the femoral head tissue,and ultimately play a role in preventing and alleviating the early hormonal necrosis of the femoral head.However, some studies have proved that high level of phosphorylated GSK-3 β It can inhibit the osteogenic differentiation of stem cells, and even accelerate the apoptosis of osteoblasts in the establishment of animal models[27-28].Therefore, accurately control GSK-3 β Phosphorylation content regulates the two signal pathways of Wnt/PI3K-AKT to maximize the proliferation and differentiation of osteoblasts and promote the repair of necrotic bone, which is of great significance for the early conservative treatment of SANFH, and is also the next research direction of the research group.

Conflict of interest

All authors of the article declare that there is no conflict of interest in the process of research and writing the article.

Author's contribution:

The experimental design is Zhou Zhengxin, the experimental guidance and evaluation are Gu Yifan and Zhu Lei, the experimental implementation is Li Zipeng and Chen Shaoqi, and the data collection and statistics are Zhu Caiyu.Li Shengchengwen and Zhou Zhengxin reviewed.