Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Yu Lixin Chen Xueqiang Wu Qunfeng Dong Weiqin Guo Songhua Yu Jian Feng Wei Yang Lei Fu Yifeng Li Junjie Luo Cheng

1 The 98th Hospital of the Chinese People's Liberation Army, Huzhou, Zhejiang 313000

2 Zhejiang University of Traditional Chinese Medicine, Hangzhou, Zhejiang 310053

3 Fuyang Traditional Chinese Medicine Orthopedics Hospital, Hangzhou City, Zhejiang Province, Hangzhou 311400, Zhejiang, China

Abstract

Objective: To observe the effect of Cistanche water extract on the healing of osteoporotic fractures in rats and to explore its mechanism. Methods: SPF-grade SD female rats were randomly divided into control group, model group, and treatment group, and modeled according to related methods. 24 hours after fracture modeling, the control group and model group were given normal saline gavage, and the treatment group was gavage with Cistanche water extract once a day. After 2 weeks and 6 weeks of intervention, blood was collected from the abdominal aorta to detect interleukins. TP (IL-1 B) and transforming growth factor-B l (TGF-8 1) levels, collect fracture specimens at the same time, perform Micro-CT scan after fixation, measure R0I callus bone volume fraction (BV/TV) and other related parameters After decalcification, dehydration, and embedding, it was made into wax blocks for use. Results: 6 weeks after the osteoporosis model was established, the bone mineral density of rats in the model group and the treatment group was lower than that of the control group (P<0.05), But there is no significant difference between the treatment group and the model group. Although the bone density of the treatment group is still lower than that of the control group after 2 weeks and 6 weeks of intervention, it is significantly higher than that of the model group (P<0.05). Micro-CT scan shows the treatment Group BV/TV, Tb. Sp, Tb. N, Tb. Th, SMI was better than the model group at 2 and 6 weeks after the intervention, and the BV/TV, Tb. Sp, SMI parameters of the control group and the treatment group were not different from those of the treatment group. Obviously, the Micro-CT parameters of the model group at 2-time points were inferior to the control group. After 2 weeks and 6 weeks of intervention, comparing the serum ILT B and TGF-P 1 levels of the 3 groups, it was found that the IL-1 P levels of the treatment group were lower than those of the other two groups at 2 weeks. At 6 weeks, the IL-1 P levels of the treatment group were higher than those of the other two groups. The control group, but still significantly lower than the model group (P<0.05); in terms of TGF-B 1 level, the treatment group was higher than the model group and the control group at 2 and 6 weeks, and there was no statistical difference between the control group and the control group. significance. Conclusion: According to the analysis of bone mineral density, Micro-CT scan parameters, and serum IL-1 B and TGF-B 1 levels, it is found that the aqueous extract of Cistanche can promote the healing of osteoporotic fractures, and its mechanism may be related to its inhibition of osteoporotic fractures. After the rat serum IL-1 B and promote the secretion of TGF-8 1, which can improve osteoporosis and promote fracture healing.

Keywords: osteoporotic fracture, Cistanche, fracture healing, rat

Osteoporosis is a disease in which the bone density is decreased, the fine structure of bone tissue is destroyed, and the fragility of bone is increased. Patients can cause fractures under slight external force in daily life. It is a common clinical disease and a frequently-occurring disease. Moreover, this type of fracture is mostly elderly patients, often associated with other diseases, and most of the fractures are comminuted, with poor stability, and sometimes slow or nonunion of fracture healing occurs, and there is a high disability and lethality rate. At present, its treatment is mainly through surgery, manual restoration, and anti-osteoporosis treatment. There is a lack of targeted treatment drugs for patients with osteoporotic fractures. Traditional Chinese medicine has a long history of treating osteoporosis and has a positive effect. Many traditional Chinese medicines have been proven to improve bones. Poorness can also promote fracture healing. Rongrong is commonly used as a representative medicine for nourishing kidney yang and has the effect of nourishing the liver and kidney. Traditional Chinese medicine believes that the kidney governs the bone, and the treatment of fractures is also inseparable from the kidney and the essence. Modern pharmacology has found that meat ash also has a certain estrogenic effect. This study intends to use bone density measurement, Micro-CT and serum interleukin-13 ( IL-10 ), transforming growth factor-P KTGF-P 1) level observation and water extract of meat hernia paste on the healing of osteoporotic fractures in rats

Cistanche

1Materials and methods

1.1 Experimental animals and reagent equipment: 52 SPF female SD rats weighing 210~250g are raised in the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. Complex iodine, Hangzhou Lonzo Medical Disinfectant Co., Ltd., batch number 20160720; Penicillin, North China Pharmaceutical Co., Ltd., batch number F3117217; IL-18 kit (batch number: 20160715), TGF-8 1 kit (batch number: 20160722); Dual-energy X-ray bone density scanner, 0STE0C0RE2; Micro-CT, Skyscan1176O

1.2 Extraction of Cistanche water extract: soak the ground cistanche (Zhejiang University of Traditional Chinese Medicine Pharmaceutical Factory) 10 times ultrapure water for 0.5 hours, frying pan decoction for 1.5 hours, repeat the extraction 2 times, and the mixed medicinal liquid rotary steamer Concentrate to a concentration of 1.5g/ml and store in a refrigerator at 4 °C for later use.

1.3 Modeling preparation: The points are as follows.

1.3. 1 Osteoporosis model: The rats were adaptively reared for 1 week before modeling and randomly divided into control group, model group, and treatment group, with 16 rats, 18 rats, and 18 rats respectively, and each group was based on the time point of 2 weeks and 6 weeks. Each was divided into two groups. Both the treatment group and the model group were anesthetized with 1% pentobarbital sodium (0.4ml/100g), the skin was anesthetized, disinfected, draped, and the skin was cut. The rat's bilateral ovaries were removed. The control group only had an incision. Do not

Removal of the ovaries

1.3. 2 Fracture model: 6 weeks after the osteoporosis model is prepared, the fracture model will be prepared. After the rats in each group are anesthetized, the bone mineral density of the middle femur is measured. , Incision, make a model of transverse fracture of the middle femur, fix it with 1mm Kirschner wire, sterilize and suture the skin of the incision

1.4 Drug intervention: X-ray detection of the femur was performed after modeling, rats that failed model preparation were eliminated, and 80,000 units of penicillin were injected intramuscularly after modeling to prevent infection. 24 hours after the operation, the treatment group was given the water extract of meat hernia paste (concentration 1.5g/ml, lg/kg, once a day). The control group and the model group were given the same volume of normal saline.

1.5 Sample collection: Rat samples were collected 2 and 6 weeks after the intervention. After 1% pentobarbital sodium anesthesia, 5-10ml of blood was collected from the abdominal aorta to prepare serum and stored in a -20B refrigerator. spare. Then the rats were sacrificed and the femoral fracture samples were collected and placed in 10% neutral formaldehyde for 72 hours.

1.6 Testing indicators: The points are as follows.

1.6. 1 Bone mineral density: All rats after the osteoporosis model were anesthetized before the fracture model, and then the bone mineral density of the middle femur was measured. The three groups of rats received samples after 2 and 6 weeks of intervention, and the samples were fixed for 72 hours and then the bone density was measured again.

1.6. 2 Micro-CT: Micro-CT scan is performed after the sample is fixed for 72 hours, with 200 pictures above and below the fracture end as the region of interest (R01), scanning angle 360°, current 555uA, voltage 42kV, filter Al 0. 2mm, resolution 8.73um, after the completion of the three-dimensional image reconstruction and analysis of the callus. The detection parameters are as follows: bone volume fraction BV/TV, bone trabecular model factor (Tb. Pf); structural model index (SMI), etc."

1.6.3 Determination of serum IL-1P and TGF-P1: Thaw the rat serum from the refrigerator at -20°C, and perform the detection step by step in accordance with the IL-13 and TGF" 1 kit instructions.

1.7 Statistical methods: use statistical analysis software SPSS.19 for processing. The experimental data are expressed in terms of mean ± standard deviation (G±s), and the bone mineral density of the three groups of rats. Micro-CT parameters JL-1 P JGF- P 1 levels are averaged by one-way analysis of variance, and pairwise comparisons between groups are used Inspection, the inspection level is P<0. 05

2 results

2.1 Bone density of the three groups: see Table 1.

Table 1 Results of bone mineral density in the middle femur of each group of rats (x±s, g/cm²)

Note: Compared with the model group, *P<0.05; compared with the control group.△ P<0.05

2.2 Micro-CT two-dimensional cross-section: the sagittal two-dimensional cross-section selects the pictures of the longitudinal axis of the femur at each time point in each group (see Figure 1), and the horizontal two-dimensional cross-section selects the 100th picture under the fracture line for comparison (Figure 2) ), it can be seen from the figure that the callus of each group gradually increased over time, but it was plastic to different degrees 6 weeks after the fracture; at the same time, it can be seen that the callus in the control group was large, and the callus cortex was thick and crawled across the fracture line after 6 weeks, while the callus in the model group There was less bone in the callus at 2 weeks, and the callus cortex was loose after 6 weeks. Although the volume of the callus in the treatment group was not large at 2 weeks, the bone mass in the callus was more than that in the model group. The callus was better shaped after 6 weeks. And the fracture line is not obvious.

Control group Model group Treatment group

Figure 1 The sagittal cross-section of the middle femur of each group

Figure 2 Horizontal section of the middle femur of each group

2.3 Micro-CT three-dimensional reconstruction: the three-dimensional reconstruction of the callus at the fractured end of the femur is shown in Figure 3. It can be seen from Figure 3 that the callus formed along the periosteum at both ends of the fracture line 2 weeks after the fracture in each group, the fracture strands in each group were still clear, especially in the model group and the treatment group, while the callus in the control group was thick and thick, mostly with plates 6 weeks after the fracture. The trabecular bones were predominant and the fracture line disappeared, while the model group was loose bones and the fracture line was fuzzy, but the treatment group had more callus bone, regular and thick trabecular bone, and the fracture line was not obvious.

Figure 3 Three-dimensional reconstruction of R0I callus at the fractured end

2.4 Micro-CT parameters: Use software to analyze the callus at the fractured end. See Table 2 for related parameters.

Table 2 Comparison of Micro-CT parameters of each group (x±s)

Control group Model group Treatment group

Note: Compared with the model group, *P<0. 05; compared with the control group. AP<0. 05

2.5 Serum IL-1 P .TGF-P 1 level: see Table 3.

Table 3 Comparison of IL-1 P JGF-0 1 levels in each group G±s, n=8)

Note: Compared with the model group, *P<0.05; compared with the control group, △P<0.05 0 3 Discussion

In recent years, the traditional Chinese medicines Yinyangdian, Drynaria fortune, and its compounds (Qing's Pills, Liuwei Dihuang Pills, etc.) have been proven in clinical practice to significantly improve osteoporosis. This type of Chinese medicine and compounds are mostly used to nourish the liver and kidney. This is consistent with the principle of nourishing the kidney and renewing the muscles in the middle and late stages of the fracture. "Ten days. The fleshy limb paste was first published in "Shen Nong's Materia Medica". , Warm in nature, has the functions of nourishing the kidney and strengthening yang, replenishing essence and marrow, nourishing blood and moisturizing dryness, and pleasing color and prolonging life.

In this study, the bone mineral density of the treatment group was higher than that of the model group after 2 and 6 weeks of intervention (P<0.05). Micro-CT scan BV/TV parameters showed the results and bone mineral density of the treatment group at two time points after the intervention The measurement results are consistent, and BV/TV is an important indicator for the evaluation of osteoporotic fracture healing. It directly reflects the bone content of the callus. This shows that Rouganrong may have the effect of promoting osteogenesis. The analysis may be effective in the meat hernia paste. The ingredients have the effect of up-regulating BMP2, and BMP2 is one of the members of the TGF-8 family. And this result is also confirmed by other parameters of the Micro-CT scan (Tb. Spjb. N, Tb. Th. SMI). Tb. Sp and SMI reflect the biomechanical properties of callus, and the values in the treatment group are significantly better than the model Group, this also further suggests that Roudongrong has the effect of improving bone loss, promoting osteogenesis, and accelerating fracture healing.

In terms of serum IL-1B and JGF-P 1 levels, after 2 weeks and 6 weeks of intervention, it was found that the IL-1P level of the treatment group was lower than the other two groups at 2 weeks, and at 6 weeks. Although the IL-1 B level of the treatment group was higher than the control group, still significantly lower than the model group (『<0.05); while the TGF-P 1 level of the treatment group was higher than the model group and the control group at 2 and 6 weeks, and there was no statistically significant difference from the control group The level of IL-1 P directly affects the differentiation and activity of osteoclasts. An increase in its level will promote bone resorption and inhibit the proliferation and differentiation of osteoblasts. JHTGF-P 1 is closely related to the proliferation, differentiation, and migration of osteoclasts. It has the effect of promoting cartilage and bone callus formation ⑵ and accelerating the shaping of bone callus. This suggests that Cistanche can improve bone loss, promote osteogenesis, and accelerate fracture healing. It may reduce the level of IL-13 in osteoporotic fracture rats and inhibit fracture. Bone cell activity is achieved; in addition, it may also be related to the fact that Cistanche can increase the level of TGF-B 1 in fractured rats, promote osteogenic differentiation and callus maturation and shaping.

In summary, based on the analysis of bone mineral density, Micro-CT scan parameters, and serum IL-1 P, TGF"1 levels, it was found that the aqueous extract of Cistanche can improve bone loss and promote healing of osteoporotic fractures. The mechanism may be related to this. It can inhibit the secretion of serum ILT 8 and promote the secretion of TGF-B 1 in rats after osteoporotic fractures, thereby improving osteoporosis and promoting fracture healing.

4 References

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