ZHU Cai-yu, ZHOU Zheng-xin, GU Yi-fan, HAN Shi-ding, XU Huan, WANG Zhuang-zhuang, ZHU Lei?

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

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

Keywords:Glucocorticoids Steroid-induced necrosis of femoral head Gubi Tongxiao granules Angiogenesis Cell apoptosis

ABSTRACT Objective: To investigate the effect of Gubi Tongxiao granules on vascular formation and cell apoptosis in the process of glucocorticoid-induced femoral head necrosis. Methods:Thirty experimental New Zealand rabbits were randomly divided into groups. Except the control group, the animal model of steroid-induced femoral head necrosis was established by lipopolysaccharide combined with glucocorticoids. After successful modeling, the experimental group was given Gubi Tongxiao granules intragastric treatment, the model and control group were given the same amount of normal saline intragastric treatment, once a day, for 8 weeks, the experimental animals were sacrificed, the bilateral femoral head of each group was taken out for hematoxylin-eosin staining, and protein CD34,CYR61 and VEGF were immunohistochemical staining localization. TUNEL staining was used to observe the apoptosis of femoral head cells. The expression levels of apoptosis proteins Bax and Bcl-2 were detected by WB. The expression of VEGF eNOs Bax and Bcl-2 genes was analyzed by RT-qPCR.Results: Gubi Tongxiao granules reduced the number of hollow bone lacunae and apoptotic positive cells(P<0.01), and up-regulated the expressions of CD34, CYR61 and VEGF in femoral head tissue. Compared with control group, the expression of pro-apoptotic protein Bax increased and the expression of anti-apoptotic protein Bcl-2, VEGF and eNOs mRAN decreased significantly in the model group (P<0.01), the opposite results were obtained in the experimental group after Gubi Tongxiao granule intervention (P<0.05). Conclusion: Gubi Tongxiao granules can improve the expression of angiogenic genes and regulate apoptosisrelated proteins in the bone tissue of rabbit model with steroid-induced necrosis of femoral head, promote angiogenic differentiation and inhibit apoptosis, and thus achieve the effect of treating steroid-induced necrosis of femoral head.

1. Introduction

Osteonecrosis of the femoral head is a common clinical refractory bone disease characterized by bone necrosis and collapse. Although the pathogenesis remains unclear, studies have shown that hormones, alcohol and trauma are common triggers [1, 2]. In recent years, with the extensive application of glucocorticoids (GCs) in anti-inflammatory and immune regulation, The incidence of steroidinduced avascular necrosis of the femoral head (SANFH) increased year by year [3].

Most scholars believe that excessive osteocyte apoptosis exists in the pathological process of SANFH, and the pathological basis of apoptosis is the interruption of blood supply caused by intravascular coagulation[4-6]. Therefore, improving the blood supply of femoral head and inhibiting apoptosis may be one of the effective targets for the treatment of femoral head necrosis. Gubi Tongxiao Granules, a traditional Chinese medicine, is an effective prescription commonly used by our research group for the treatment of femoral head necrosis, and combined with surgery can significantly improve the prognosis of hip preservation[7, 8]. In order to further prove the protective effect of Gubi Tongxiao Granules, this experiment started from the aspect of vascularization, studied the relationship between vessel formation and cell apoptosis in the process of femoral head necrosis in SANFH rabbit model, and explored the possible mechanism of Gubi Tongxiao granules in the treatment of SANFH.

2. Materials and Methods

2.1 The experimental materials

2.1.1 Thirty clean grade maleNew Zealand rabbits, weighing 3.5-4.0 kg, were purchased from Anhui Animal Experimental Center (License NO. SYXK (Anhui)2020-001), Animal qualification NO.202213656. The experimental protocol was approved by the Animal Experiment Ethics Committee of Anhui University of Traditional Chinese Medicine (Ethics number: AHUCM-rabbits-2022021).

2.1.2 Experimental drugs

Gubi Tongxiao granules (15 g salvia miltiorrhiza, 12 g paeoniae paeoniae, 10 g each of Chuanxiong, Angelica sinensis, continuous tablets and Herba epimedium, 6g each of Tubiecia and licorice,cinnamon 4G) and methylprednisolone sodium succinate for injection (40 mg/ branch, Pfizer Manufacturing Belgium NV Pharmaceutical, batch number: DH6371) were provided by the Pharmacy of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.

2.1.3 Lipopolysaccharide

(Biosharp, Lot No.:21333172); TUNEL apoptosis detection Kit(Baisai, batch No.210419L1-1); Tissue lysate (Shanghai Biyuntian Biology, Batch No.:042121210708); Sds-page gel rapid preparation kit (Soleibo Bio, Lot No.: CR2110087); CD34, CYR61 and VEGF antibodies (Shenyang 10000 organisms, lot No.:NO3073380,NO3011435, NO3070962); Antibodies against β-actin, Bax and bcl-2 were purchased from Bioss (lot No. AH11286487, BJ10163878,BJ11026965). Cell RNA rapid extraction Kit (Guangzhou Meiji Biology, batch No.:RJH19-01); Reverse transcription and real-time PCR kit (Shanghai Xinbei Biological, batch No.:F2987, F3528);Tissue dehydrator, embedding machine, paraffin slicer (German Microm company, model:STP120, AP280-2, HM355S); Inverted fluorescence microscope (Olympus Biology, Model: CX53); WB gel imaging analysis system (Bio-Rad, model: ChemiDoc); Real-time PCR instrument (ABI Company, model: QuantStudio 5).

2.2 The experimental method

2.2.1 Animal Model

Replication and intervention Thirty New Zealand rabbits were randomly divided into control group, model group and experimental group, with 10 rabbits in each group. The model group and experimental group were injected with endotoxin at 10 μg/kg gluteal muscle, and 24 h later, the left and right gluteal muscles were alternately injected with 20 mg/kg methylprednisolone sodium succinate for 3 times with an injection interval of 24 h.After successful modeling, the experimental group was converted to the equivalent dose of human and animal [rabbit equivalent dose=(human drug dose)/60 kg×3.3], and the experimental group was gavaged with 4.5 g/kg Gubi Tongxiao granules, while the model group and the control group were given the same amount of normal saline. After 8 weeks, the experimental animals were sacrificed and bilateral femoral heads were removed. The samples were stored in 4% paraformaldehyde and -80℃ refrigerator for later use.

2.2.2 Observation of Histopathological changes by HE staining

The femoral head samples fixed in paraformaldehyde were removed and decalcified with 10% formic acid solution. After the completion of the decalcified, paraffin embedding, sectioning, roast,after the biopsy for dewaxing hydration, then in turn to carry on the wood grain, eosin staining, using different concentrations of ethanol dehydration step by step, xylene fully infiltrates use neutral gum after sealing piece, the last slice was observed and photographed under light microscopy, calculation of empty pit rate.

2.2.3 Some sections were selected for immunohistochemical

Staining of CD34, CYR61 and VEGF. After xylene dewaxing and hydration, thermal repair was performed. After treatment with 3%hydrogen peroxide deionized water for 5 min, the primary antibodies were incubated overnight at 4℃. After 20 min, the sections were rinsed with PBS again, and the sections were observed under the microscope after DAB color development, and the reaction was terminated when the target protein was brown-yellow, and the background was not colored. Then hematoxylin was used for counterstaining, rapid differentiation, washing, bluing with PBS,dehydration and transparent, and the sections were sealed for observation under the microscope and images were taken.

2.2.4 Detection of cell apoptosis in femoral head area by TUNEL Method

The femoral head samples were prepared into paraffin tissue sections. After dewaxing at room temperature, the tissue was permeated with 20 μg/mL proteinase K solution, and the TUNEL reaction mixture was added after fully rinsing with PBS. The sample was evenly covered with the reaction solution with a cover glass and placed flat in the wet box. After incubating at 37℃ for 2 h, the reaction solution was removed, and the sample was moistened and washed twice with PBS. The sections were washed with 0.1% Triton X-100 buffer containing 5 mg/mL BSA to reduce background,and apoptosis was subsequently observed under a fluorescence microscope.

2.2.5 The femoral head samples placed in the refrigeratorAt -80℃ were ground in a tissue grinder, and the total protein of bone tissue was extracted. The protein concentration was determined by BCA method. Gel electrophoresis was performed according to 10μl of sample and 5 μl of marker in each well. In semi-wet method,the protein was transferred to PVDF membrane and blocked with 5% skim milk for 2 h. The primary antibody was incubated at 4℃overnight with a shaker. The second antibody was incubated with a shaker for 2 h at room temperature the next day. After that, the membrane was fully washed with TBST solution to reduce the background. The PVDF membrane was infiltrated into the developer and placed in the Western blotting system to detect the Image.

2.2.6 RT-QPCR Detection of VEGF, eNOs, Bax and Bcl-2 mRNA Expression

Total RNA was rapidly extracted from femoral head tissue samples after low temperature grinding by double-column centrifugation and its purity was measured. According to the known sequences of target genes in GeneBank (see Table 1). According to the instructions of the reverse transcription and real-time PCR kit, the reaction program was set with β-actin as the internal reference, and the total RNA was put into the Q-PCR instrument for reverse transcription,amplification and quantitative detection. The results were statistically analyzed by 2-ΔΔCtmethod.

Tab 1 Primer sequences of each gene

2.2.7 All experiments were repeated three timesAnd the experimental data were analyzed and processed by SPSS 25.0 statistical software. Measurement data were described by mean±standard deviation(±s ), and one-way analysis of variance was used to compare sample means among multiple groups. In this experiment, P < 0.05 was used to indicate statistical significance.

3. Results

3.1 Histopathological results of femoral head of rabbits in each group

After 8 weeks of SANFH model replication, the trabecular bone area of rabbits in the model group was significantly reduced, there were severe stenosis or fracture, the types of cells in the medullary cavity were reduced, and the number of empty bone lacunae was significantly increased compared with the control group (P<0.01).After the intervention of Gubi Tongxiao granules, the rabbit medullary cavity cells in the experimental group were more diverse and abundant, the trabecular bone structure was more complete than that in the model group, and the empty bone lacunae rate was significantly lower than that in the model group (P<0.01) (see Figure 1 and Table 2).Group Empty bone lacuna rate(%)(%)Note: Compared with the control group,**P<0.01; Compared with the model group,##P< 0.01

Tab 2 Comparison of hollow bone lacunae rate of rabbit femoral head tissues in each group (n=3. ±s )

Tab 2 Comparison of hollow bone lacunae rate of rabbit femoral head tissues in each group (n=3. ±s )

Control 5.401±0.226 Model 19.263±0.960**Experience 9.414±0.424##F 397.398 P P<0.01

3.2 The results of immunohistochemical

staining showed that CD34, CYR61 and VEGF proteins were positively expressed in different degrees in the pathological sections of femoral head. The results showed that the protein levels of CD34,CYR61 and VEGF in the model group were significantly lower than those in the control group. Compared with the model group,the results of immunohistochemical localization staining in the experimental group were effectively improved, and the expression of vasculogenesis related factors in the femoral head tissue was increased, but there was still a certain gap compared with the normal group (Figure 2).

3.3 Apoptotic cells in femoral head area of rabbits in each group

Were stained with TUNEL staining and the apoptotic nuclei were brownish yellow as single cells or scattered, mostly located at the junction of diseased tissues and normal tissues. Compared with the control group, the number of apoptotic cells in the model group increased and the nuclei were darker, while the apoptosis of femoral head cells in the experimental group was significantly improved after treatment with Gubi Tongxiao granules (P < 0.01) (Figure 3, Table 3).

Tab 3 Comparison of cell apoptosis in femoral head area among all groups(n=3.±s )

Tab 3 Comparison of cell apoptosis in femoral head area among all groups(n=3.±s )

Note: Compared with the control group, **P<0.01; Compared with the model group, ##P< 0.01

Group Apoptosis rate(%)Control 3.703±0.183 Model 20.113±0.679**Experience 8.970±0.349##F 1025.568 P P＜0.01

3.4 The expression of Bax and bcl-2 protein in femoral head tissue

Was shown by Western blot results. The expression of proapoptotic protein Bax in the model group was significantly increased, while the expression of anti-apoptotic protein Bcl-2 was inhibited (P < 0.01). The protein expression levels of Bax and Bcl-2 in the experimental group were opposite to those in the model group(P < 0.01) (see Figure 4, 5, Table 4).

Tab 4 The expression level of target protein in femoral head tissue of each group(n=3.±s )

Tab 4 The expression level of target protein in femoral head tissue of each group(n=3.±s )

Group Bax Bcl-2 Control 0.523±0.016 1.285±0.020 Model 2.018±0.029 0.627±0.023 Experience 1.073±0.035 0.988±0.035 F 2235.454 445.162 P＜0.01 ＜0.01

3.5 Compared with the control group

the mRNA expression of VEGF, eNOs, Bcl-2 and Bax in the femoral head tissue of the model group was significantly decreased,while the expression of pro-apoptotic gene Bax was increased(P<0.01). After the intervention of Gubi Tongxiao granules, the mRNA expression of Bax in the experimental group was inhibited,and the mRNA expression levels of VEGF, eNOs and Bcl-2 were significantly increased compared with those in the model group(P<0.01) (see Figure 6 and Table 5).

Tab 5 mRNA expression level in femoral head tissue of each group(n=3. ±s)

Tab 5 mRNA expression level in femoral head tissue of each group(n=3. ±s)

Group VEGF eNOs Bcl-2 Bax Control 0.957±0.087 1.271±0.065 1.002±0.021 1.008±0.065 Model 0.456±0.070 0.697±0.119 0.585±0.064 1.549±0.107 Experience 0.786±0.038 0.954±0.044 0.924±0.020 1.194±0.061 F 42.001 36.620 89.188 35.056 P <0.01 <0.01 <0.01 <0.01

4. Discuss

At present, among the several pathogenesis of osteonecrosis of the femoral head, GCs induces osteoblast apoptosis, leading to femoral head bone mass reduction and even collapse, which is still widely concerned[9, 10]. Clinical treatment options for SANFH include nonsurgical treatment, hip preservation surgery and hip replacement,among which total hip replacement is also considered as the last resort for SANFH patients[11]. Literature has shown that[5, 12], with the emergence of SARS and COVID-19, the incidence of GCSinduced AVascular necrosis of the femoral head gradually increases and the age of onset tends to be younger, which means that most young and middle-aged patients need to consider the durability of postoperative artificial hip prosthesis and the feasibility of revision.So anything that can delay or avoid hip replacement makes sense.

Although there are many etiology and pathogenesis of femoral head necrosis, their common feature is that the interruption of local blood supply of femoral head leads to osteocyte ischemia, necrosis or apoptosis, and the consequent body repair reaction, which leads to the pathological process of bone mass reduction and even femoral head collapse[2, 13]. Studies have shown that long-term or high-dose use of GCs can damage vascular endothelial cells, cause intravascular hypercoagulation, resulting in interruption of femoral head blood supply. In addition, under the stimulation and induction of GCs,the intracellular apoptosis pathway is activated, which eventually causes extensive apoptosis of osteocytes[14, 15]. Li et al.[16] believe that ischemia, necrosis and collapse are different pathological stages in the course of osteonecrosis of the femoral head, and ischemia is the first and reversible process, while the latter two are irreversible.As important markers and regulators of vascular formation, CD34 and CYR61 can not only accelerate the adhesion, migration and aggregation of microvascular endothelial cells to form blood vessels,but also regulate hematopoietic stem cells, promote cell proliferation and differentiation, and achieve vascular regeneration[17, 18]. VEGF is a highly specific pro-vascular endothelial cell growth factor,which can promote the migration, proliferation and angiogenesis of vascular endothelial cells[19]. Studies have found that VEGF and eNOs participate in bone development through vascular formation and accelerate bone vascularization through vascular proliferation,which is of great significance for clinical anti-osteoporosis[20].

Contemporary physician Liu Bailing believes[21] that the occurrence of femoral head necrosis is mainly caused by liver and kidney deficiency, qi stagnation and blood stasis as the key factors,and positive deficiency and evil invasion as the important causes.Therefore, in the treatment of traditional Chinese medicine, most of the emphasis on tonifying kidney Yang, strengthening muscles and bones, promoting blood circulation and removing blood stasis and other treatment methods. The research shows that the traditional Chinese medicine of tonifying kidney and activating blood in the treatment of osteonecrosis of femoral head not only has distinct characteristics, but also has definite curative effect. Bao Jiamin et al.[22] found that total saponins of Panax notoginseng can upregulate the expression of VEGF and receptor proteins in SANFH model rabbits, promote angiogenesis, and play a positive protective role in GCS-induced femoral head necrosis. Chen et al.[23] found that Taohong Siwu decoction could effectively delay the process of SANFH by regulating the expression of VEGF in animal models and significantly inhibiting cell apoptosis. Xu Huihui et al.[24]found through experiments that platelet-rich plasma combined with Taohong Siwu decoction could up-regulate the expression of VEGF in femoral head tissue of SANFH model rats, improve local blood supply of femoral head, and promote the repair of necrotic bone. In this experiment, after successful replication of the SANFH rabbit model, HE staining showed obvious stenosis or fracture of the trabecular bone in the femoral head of the model group, and increased adipocytes in the medullary cavity with signs of fusion.After the treatment of Gubi Tongxiao granules, the trabecular bone area increased and the rate of empty bone lacunae decreased significantly. The expression levels of CD34, CYR61, VEGF and eNOs were significantly increased compared with the model group.

Studies have found[25, 26] that the regulation of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 through the mitochondrial pathway may be a potential mechanism to promote cell survival. In the mitochondrial pathway, Bcl-2 family proteins regulate the release process of mitochondria-related apoptotic factors, and the ratio of Bax/Bcl-2 determines the survival status of cells[27]. When cells receive apoptosis signals induced by hormones, the expression of Bax in the cytoplasm is up-regulated and translocated to the outer mitochondrial membrane, which promotes the over-opening of mitochondrial membrane permeability transport pores and the release of other apoptotic proteins, and then the formation of apoptotic bodies and the induction of largescale apoptosis of osteoblasts[28]. Bcl-2, which is normally located in the outer membrane of mitochondria, continuously relocates the translocated Bax back into the cytoplasm, thereby promoting cell survival. However, under GCs exposure, Bcl-2 expression decreases, its ability to stabilize mitochondrial membrane function decreases, cell apoptosis increases, and eventually leads to bone loss and even osteonecrosis[29]. In this experiment, the combined induction of LPS and GCs promoted increased apoptosis of femoral head, necrosis and collapse of femoral head in different degrees in SANFH rabbit model. Western blot results showed that GCs induced high expression of pro-apoptotic protein Bax and low expression of anti-apoptotic protein Bcl-2 in bone tissue, resulting in increased apoptosis of osteocytes. In the experimental group, the apoptosis of osteocytes in femoral head tissue was improved, and the abnormal expression of Bax and Bcl-2 mRNA was also reversed by Gubi Tongxiao granules.

In conclusion, there are abnormal local blood supply and excessive apoptosis of osteocytes in the pathological process of GCs-induced osteonecrosis of the femoral head. Gubi Tongxiao granules has a significant protective effect on the ischemic apoptosis of osteocytes in GCs environment, which can up-regulate the expression of CD34,CYR61 and VEGF, promote angiogenesis, and facilitate the repair of necrotic bone. At the same time, Gubi Tongxiao granules can stabilize the function of mitochondrial membrane and reduce the apoptosis of osteocytes induced by GCs by regulating Bax/Bcl-2 complex, so as to play a role in treating SANFH.

Conflicts of interest: The content of this article does not involve relevant conflicts of interest.

Author contributions: Zhu Lei designed the experiment, Zhou Zhengxin and Han Shiding supervised the experiment, Zhu Caiyu,Gu Yifan and Wang Zhuangzhuang conducted the experiment, Xu Huan, Zhu Caiyu Chengwen, and Zhu Lei reviewed the data.