Experimental Study on the Repair Effect of Xianlinggubao Capsule on Osteoporotic Vertebral Compression Fracture in Rabbits

Lihong GUO, Lizhu LIU, Xi WANG, Heng LIAO, Jingping MU

Center for Sleep Psychosomatic Medicine, Taihe Hospital of Shiyan City (Affiliated Hospital of Hubei University of Medicine), Shiyan 442000, China

Abstract [Objectives] To observe the effect of Xianlinggubao Capsule on osteoporotic vertebral compression fracture (OVCF) in rabbits and the influence mechanism of the repair of fractures. [Methods] Female June age 30 rabbits were randomly divided into control group, model control group and Xianlinggubao group. After bilateral ovariectomy, the model control group and Xianlinggubao group were injected with dexamethasone continuously for 4 weeks, and then the OVCF compound model was established by surgery. The Xianlinggubao group was treated with Xianlinggubao at a dose of 300 mg/(kg·d) for 60 d, while the blank control group and the model control group were treated with the same amount of normal saline for 60 d. The number of blood vessels and the expression of bone morphogenetic protein-2 (BMP-2) were detected by immunohistochemical staining and the bone mineral density (BMD) in the callus of the third lumbar fracture area of rabbits was measured. The content of serum phosphorus (P), alkaline phosphatase (ALP) and total calcium (TCa) in rabbit venous blood were measured by automatic biochemical analyzer. The content of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in rabbit venous blood were measured by ELISA kit. [Results] The number of blood vessels and the expression of BMP-2 in the callus of the third lumbar fracture area of rabbits was high in Xianlinggubao group, the content of serum P, ALP, TCa, VEGF and PDGF was obviously increased, BMD was obviously increased, the bone microstructure of the third lumbar vertebrae fracture area of rabbits was basically restored. Compared with the model control group (P<0.05), the difference was statistically significant. [Conclusions] Xianlinggubao Capsule can increase calcium and phosphorus deposition, promote the formation of blood vessels in the fracture area of OVCF in rabbits, and have a strong repair effect on OVCF in rabbits.

Key words Rabbit, Xianlinggubao Capsule, Osteoporosis, Compound model of vertebral compression fracture, Repair

1 Introduction

Osteoporotic vertebral compression fracture (OVCF), as a lumbar vertebral compression fracture caused by osteoporosis, is one of the most serious complications of osteoporosis[1]. OVCF occurs in L1-L5 of vertebral body, and the elderly are at high risk, among which the elderly women are more likely to develop this disease[2]. The pathological process of OVCF healing is basically consistent with that of normal fracture, that is, inflammatory reaction-hematoma organization stage-original callus formation stage-callus reconstruction and shaping stage. However, due to the imbalance of calcium and phosphorus metabolism in osteoporosis patients, the decrease of bone calcium deposition leads to the decrease of bone mass, bone density and strength are reduced, and bone fragility is increased, which is easy to cause compression fracture due to external force[3]. The incidence of OVCF is increasing year by year, posing the most serious threat to the health of the elderly. Therefore, timely correction of calcium and phosphorus metabolism and prevention of osteoporosis are the key to preventing porous fracture and fracture recurrence. At present, there have been some researches on the effect of Xianlinggubao on fracture healing, but the mechanism of its effect on early fracture healing after osteoporosis is not clear. In this study, Xianlinggubao Capsule was used to treat OVCF in ovariectomized rabbits, and the effects of Xianlinggubao Capsule on many related factors in the process of fracture healing in OVCF model were observed. The research results are summarized in the following.

2 Materials and methods

2.1 Animals and groups30 female 6-month-old rabbits, weighing between 2 500 and 2 800 g, were purchased from the animal room of Hubei University of Medicine. They were randomly divided into three groups: blank control group, model control group and Xianlinggubao group, with 10 rats in each group.

2.2 Instruments and reagentsXianlinggubao Capsule (Guizhou Tongjitang Pharmaceutical Co., Ltd., batch number: C12001226041); VEGF and PDGF ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd.); chloral hydrate (Yangzhou Aoxin Additives Factory, batch number: 201708023); dexamethasone (Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., batch number: H5021200514); Media Cybernetics Image Analysis System (Media Cybernetics of the United States); WD-240vet Automatic Biochemical Analyzer (Jilin Weier Medical Devices Co., Ltd.); X-ray bone densitometer (Tianjin Shenghong Medical Devices Co., Ltd.); SG-51 Positive Metallographic Microscope (Shanghai Optical Instrument Factory).

2.3 Establishment of OVCF compound animal model and treatmentOVCF was established by continuous injection of dexamethasone for 4 weeks after bilateral ovariectomy in rabbits.

(i) Ovariectomy method[4]. First, the rabbits in the model control group and Xianlinggubao group were anesthetized and fixed by intraperitoneal injection of 10% chloral hydrate (0.35 g/kg), and the abdominal hair was removed and disinfected by sodium sulfide. The left abdominal skin (about 1 cm) was cut longitudinally at L1-L5, the left abdominal muscles were separated and the peritoneum was cut. After finding the ovary, the fallopian tube was ligated with silk thread and the ovary was separated and removed, and then the right ovary was separated and removed by the same method. Bilateral peritoneum, muscle, fascia and skin were sutured and disinfected. The above operations were performed in a bacterial-free environment without antibiotics after operation, and the surgical wounds healed one week later.

(ii) Glucocorticoid induced osteoporosis method[5]. After ovariectomy, dexamethasone was injected intramuscularly for 4 weeks, and the dosage was 1 mg/(kg·d) once a day.

(iii) Osteoporotic vertebral compression fracture surgery: After ovariectomy, the rabbits in the model control group and Xianlinggubao group were treated with operation and forced exercise to make vertebral compression fracture (OVCF compound animal model)[6]. The rabbits were fixed in the right lying position, depilated and disinfected, then sterile towel was laid, and L3 was located accurately. The skin of the right vertebral body was cut longitudinally, the fascia, anterior vertebral muscle and intervertebral disc were separated, the anterior edge of the vertebral body was exposed and cut, and then it was sutured. OVCF compound animal model can be established by forced exercise for 30 min every day for 4 weeks after operation.

(iv) Treatment: Xianlinggubao Capsule was prepared into 10% suspension. The Xianlinggubao group was treated with Xianlinggubao intragastric administration at a dose of 300 mg/(kg·d) for 60 d, while the blank control group and the model control group were treated with the same amount of normal saline for 60 d without any treatment[7-8]. This experiment takes a long time, and intragastric administration can be done once a day.

2.4 Test and methods of related indexes(i) After treatment, about 1 mL of rabbit tail vein blood was taken. The content of phosphorus (P), alkaline phosphatase (ALP) and total calcium (TCa) in rabbit venous blood was detected by automatic biochemical analyzer, and the content of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in rabbit venous blood was measured by enzyme-linked immunosorbent assay kit.

(ii) Measurement of bone mineral density in rabbits. The bone mineral density (BMD) of the third lumbar vertebrae fracture area was measured by X-ray bone densitometer after the treatment.

(iii) Pathological examination. Hematoxylin-eosin staining was used to detect the number of blood vessels. The rabbits were killed, the callus tissue of the third lumbar vertebrae fracture area was decalcified, embedded in paraffin, stained with hematoxylin-eosin, and the number of new vessels in callus tissue was observed under 1：100 visual field with SG-51 positive metallographic microscope. Three visual fields were randomly selected for counting and the number of new vessels was averaged.

(iv) Expression of bone morphogenetic protein 2 (BMP-2). Immunohistochemical staining was used to detect the expression of BMP-2 in callus tissue of the third lumbar vertebra fracture area. The callus tissue specimens of the third lumbar vertebrae fracture area were sliced, dewaxed, then dripped with peroxidase blocking solution, and then incubated for 10 min. The animal BMP-2 antibody was added dropwise and digested overnight at 4 ℃. Then it was rinsed with PBS solution for 1 min, and incubated for 10 min after dropping second antibody. Once again, it was washed with PBS solution and incubated with streptomycin-avidin-peroxidase solution for 10 min. After color development with DAB, it was washed, re-dyed and sealed. The absorbance value (ODvalue) of positive cells was measured by Image-Pro Plus professional image analysis software.

3 Results and analysis

3.1 Comparison of the number of new vessels, BMP-2 and BMD in callus tissue of the third lumbar vertebra fracture area in rabbitsIt can be seen from Table 1 that the number of new vessels in callus tissue of the third lumbar fracture area in the model control group was small, and BMD was lower than that in the blank control group (P<0.05), and the difference was statistically significant. BMP-2 was expressed in trabecular bone and osteoblast cytoplasm in model control group, but the difference was not statistically significant compared with that in blank control group (P>0.05). Compared with the model control group, the number of blood vessels and the expression of BMP-2 were higher and BMD was higher in Xianlinggubao group (P<0.05).

Table 1 Comparison of the number of new vessels, BMP-2 and BMD in the third lumbar fracture area of rabbits in three groups

3.2 Comparison of serum detection results among three groups of rabbits after treatmentIt can be seen from Table 2 that the content of serum P, ALP, TCa and PDGF in the model control group decreased significantly, while VEGF increased, suggesting that the difference was statistically significant compared with the blank control group (P<0.05). The content of serum P, ALP, TCa, VEGF and PDGF in XLGB group was significantly higher than that in model control group (P<0.05).

Table 2 Comparison of serum test results of three groups of rabbits after treatment n=10)

4 Discussion

Xianlinggubao Capsule, which is included in theNationalEssentialDrugsCataloguein 2009, is an over-the-counter traditional Chinese medicine preparation for osteoporosis, fracture and other orthopaedics. The capsule contains traditional Chinese medicine components such asFructuspsoraleae,epimedium,RehmanniaglutinosaLibosch. andDipsacusasper. The capsule has the effects of promoting blood circulation, dredging collaterals, strengthening tendons and bones, nourishing liver and kidney,etc.[9-10]. A large number of clinical studies have shown that Xianlinggubao has a significant preventive and therapeutic effect on osteoporosis, and is used to treat and prevent osteoporosis, osteoarthritis, fracture, aseptic necrosis of bone and other orthopedic diseases. It is the first choice of traditional Chinese medicine preparation for preventing and treating osteoporosis[11]. In addition, basic medical research shows that Xianlinggubao Capsule can significantly improve the bone mass of osteoporotic fracture rats, increase the bone density of ovariectomized rats, prolong the fracture healing time, and has the functions of reducing blood lipid, improving local tissue blood flow of osteoporotic fracture, promoting calcium and phosphorus metabolism, and increasing calcium deposition to accelerate the transformation from cartilaginous callus to bony callus[12-13].

The organs of the elderly are in decline, especially in postmenopausal elderly women. Most of the patients have decreased bone matrix and bone calcium due to the disorder of hormone level and calcium and phosphorus metabolism in the body, and the bone absorption activity is enhanced, while the osteoblast activity is weakened, and the bone mass is gradually lost, eventually leading to osteoporosis[14-15]. Due to the gradual loss of bone mass and the decrease of BMD after osteoporosis, the bone strength is obviously reduced. Any part of the body is prone to fracture under the action of external force, and the third lumbar vertebra is most prone to compression fracture (i.e.OVCF). Therefore, the degree of osteoporosis is the main risk factor for osteoporotic fracture and fracture recurrence[16]. Studies have shown that PDGF can obviously promote the intramembranous osteogenesis during the healing process of OVCF, and it can induce mesenchymal stem cells to differentiate into osteoblasts and promote the proliferation of osteoblasts in the fracture area of OVCF. Therefore, the content of PDGF in serum represents the proliferation activity and differentiation degree of osteocytes[17]. VEGF is the strongest important factor to promote angiogenesis, and it can promote angiogenesis in fracture area[18]. BMP-2 can stimulate the differentiation of mesenchymal stem cells, promote the differentiation of mesenchymal stem cells into osteoblasts, and regulate the formation, development and reconstruction of bone by regulating the differentiation of osteoblasts into osteoblasts, so its high expression is beneficial to increasing bone mass and promoting fracture healing[19-20]. ALP, secreted by osteoblasts, can hydrolyze pyrophosphate and phosphate in the process of osteogenesis. Osteoblasts become active from rest during osteoporotic fracture. At this time, bone resorption is hyperactive, and ALP in serum is increased due to the increase of compensatory bone formation, which reflects the degree of bone formation in the process of bone turnover to a certain extent[21].

Based on the successful establishment of rabbit osteoporosis model, the OVCF compound animal model was established by surgery, and then Xianlinggubao Capsule was given by intragastric administration for 60 d. The results showed that the number of blood vessels and the expression of BMP-2 were high in the callus tissue of the third lumbar vertebrae fracture area, the content of serum P, ALP, TCa, VEGF and PDGF was significantly increased, BMD was significantly increased, and the bone microstructure of the third lumbar vertebrae fracture area of rabbits was basically restored. These results suggest that Xianlinggubao Capsule can promote the healing of OVCF, and its mechanism may be that Xianlinggubao Capsule can promote the synthesis and secretion of VEGF and PDGF, and promote the angiogenesis of callus tissue in fracture area, which is beneficial to improving blood circulation, increasing blood flow and promoting metabolism in fracture area. In addition, Xianlinggubao Capsule can also enhance the expression of BMP-2, which is beneficial to the differentiation of mesenchymal stem cells into osteoblasts and increase of the formation, development and reconstruction of bone. By increasing the secretion of ALP, we can hydrolyze pyrophosphate and phosphate in the process of osteogenesis, inhibit the hyperabsorption of bone during osteoporotic fracture, regulate the metabolism of P and TCa, promote the deposition of bone calcium, increase BMD and enhance bone strength.

In a word, Xianlinggubao Capsule can increase calcium and phosphorus deposition, promote the formation of blood vessels in the fracture area of OVCF in rabbits, and has a strong repair effect on OVCF in rabbits.