Abstract Objective To investigate the effect of gecko extract on the prevention and treatment of osteoporosis. Methods The animal model of osteoporosis was established by removing both ovaries of healthy female SD rats, and 0.2g/kg of gecko extract was administered by gavage after surgery. 6 months after surgery, bone tissues of rats in each group were taken and tested for bone density value, bone calcium content, maximum stress, maximum strain, elastic modulus, bone cortical thickness and other indexes. Results There were significant differences in all indexes between the sham-operated group and the model group, indicating that the animal model of osteoporosis was prepared successfully. The maximum stress, maximum strain, elastic modulus, bone density value, bone calcium content and bone cortical thickness of bone tissue of rats in the prevention and treatment groups were significantly higher than those in the model group, and the differences were statistically significant (P<0.01); the diameter of bone marrow cavity was reduced in the prevention and treatment groups (P<0.01, P<0.05); there was no significant difference between the indicators of bone tissue of rats in the prevention and treatment groups compared with each other. Conclusion Gecko extract can increase bone mass and bone strength, and has a certain preventive and curative effect on osteoporosis. Liu Yishan, Department of Integrative Orthopedic Treatment, Air Force General Hospital
[Keywords] Osteoporosis; webless gecko; rats
[I.C.C.] R68 [I.D.] A [Article ID] 100021824(2006)020174204
Experimental study of gecko swinhonis gunther preventing and curing osteoporosis
LIU Yi-shan1 ,REN Dong-qing2,GUO Wei1,CI Yan-feng3
(1. PLA Center of Integrated Traditional and Western Medicine for Manipulative Orthopedics, General Hospital of Air Force, Beijing 100036,China; 2. Department of Radiation Medicine, Faculty ABSTRACT: OBJECTIVET To research the function of gecko swinhonis gunther prevents in gland curing steoporosis. METHODS 24 healthy female SD rats were randomly divided into 4 groups: control group, model group, prevention group and therapy group. Except for control group, the rest 3 groups were all operated on variectomy. Rats in the prevention group were dosed 02g/kg per day from stomach in the third day after operation. Rats in the therapy group were dosed 0.25g/kg per day from stomach in the third month after operation. Rats in the control group and model group were dosed equal NS. After 6 months, all rats were executed to measure indexes. RESULTS The maximum stress, maximum strain, lactic modulus, the changes of bone mineral density of the whole bone and lumbar vertebra, the content of bone calcium, the cortex of bone thickness of prevention group and therapy group were obviously higher than that of model group, compared to control group, there were no significant differences. The marrow cavity diameter we Keywords: osteoporosis; gecko swinhonis gunther; bone density; rats Osteoporosis (OP) is a common metabolic disease that poses a serious health risk to middle-aged and elderly people. The prevalence of osteoporosis has jumped to the seventh place of common diseases and diseases in the world, with about 200 million people suffering from osteoporosis. Osteoporosis is a metabolic disease characterized by reduced bone mass, structural degeneration of bone tissue, decreased bone strength, increased bone fragility, and increased risk of fracture. To date, osteoporosis is commonly treated with estrogen replacement therapy as its main treatment, but there are many risks such as breast cancer [1]. Therefore, the search for ideal and highly effective safety drugs has become a hot topic of research. In this study, we propose to investigate the effect of Chinese herbal medicine Gecko extract on the prevention and treatment of osteoporosis by using a model of osteoporosis in desiccated female rats. 1 Materials and Methods 1.1 Materials Twenty-four healthy female grade II SD rats, (220±15) g, 8 weeks old, were provided by the Animal Center of the Fourth Military Medical University. The rats were randomly divided into 4 groups according to their body weight i.e. sham-operated group, model group, prevention group and treatment group with 6 rats in each group after 1 wk of acclimatization feeding. qdr4500w dual line x-ray bone densitometer (HOLOGIC, USA), MTS-858 biomechanical test machine (MT Systems, USA), gecko extracts were prepared in our laboratory. 1.2 Osteoporosis model preparation Referring to the literature [2], the method was slightly improved. 1% pentobarbital sodium (50 mg/kg, ip) was used to anesthetize the rats intraperitoneally, and the lower abdomen of each group was incised under aseptic conditions. The rats in the remaining groups were freed and bilateral ovaries were removed, and the abdominal wall was sutured. The rats in the sham-operated group had only a small amount of adipose tissue removed from the free ovaries and then the abdomen was closed immediately, while the rest of the rats in each group had both ovaries removed, the fallopian tubes ligated and the wounds sutured.
The rats were kept for 5 d postoperatively with intramuscular injection of gentamicin sulfate for anti-infection and 6 rats per cage; the feeding temperature was 12~25℃ and humidity was 50%~80%. 1.3 Gecko extract and gavage administration Gecko (Gecko swinhonis Gunther) of the dried body, water decoction alcoholic sedimentation method to prepare the extract, the extract was freeze-dried to remove water, according to each bottle of 2g of the original medicine, 4 ℃ storage, diluted with water for injection before use. The rats in the prevention group were given the drug at 6 d postoperatively, and the rats in the treatment group were given the drug at 3 mo postoperatively. Both groups were given 1 ml of gecko extract by gavage at 0.2 g/kg body weight per day, and the rats in the sham-operated and model groups were given the same amount of water for injection by gavage at 6 d postoperatively. The rats in each group were executed at 6mo after surgery and tested for each index. 1.4 Bone mineral density (BMD) was measured in the whole body of rats and their 2~4 lumbar vertebrae using a dual-line X-ray bone densitometer. 1.5 Biomechanical measurement The left femur of the rat was soaked in 70% ethanol and the soft tissue was removed by wiping it with gauze. 1.6 Measurement of bone calcium content Take the right humerus of rats, wipe off the soft tissue, burn it to ash in an electric furnace crucible at 750℃, dissolve it in 40ml solution of 6mol/L hydrochloric acid, and determine the bone calcium content by EDTA complex titration method. 1.7 Bone cortical thickness measurement The right femur was dissected, the soft tissue was removed, the bone tissue was sawed in the upper middle 1/3 of the femur, and the bone cortical thickness and the diameter of the bone marrow cavity were measured with electronic vernier calipers. 1.8 Statistical treatment Data were expressed as mean ± standard deviation, and the analysis of variance (ANOVA) in SPSS13.0 software was tested, and the data were tested for chi-square with Levene’s test, and the chi-square was then tested for mean two-by-two comparison with LSD, and if the chi-square was not chi-square, the non-reference Mann-Whitney Test was used for test. 2 Results As shown in Table 1, the whole-body BMD, lumbar spine BMD and bone calcium content of the rats in the sham-operated, preventive and treatment groups were significantly higher than those in the model group, and the differences were statistically significant (P<0.01). Table 1 Comparison of lumbar spine and whole body BMD values and bone calcium content of rats in each group (n=6, ±s) subgroups whole-body bone mineral density values L2~L4 BMD Bone calcium content (mg/g) model group 0.1377±0.0056 0.148±0.004 129.3± 5.7 sham-operated group 0.1682±0.0034 b 0.170±0.004 b 169.5±12.3 b Prevention group 0.1587±0.0014 bd 0.165±0.005 bc 157.0± 13.7 b Treatment group 0.1579±0.0022 bd 0.163±0.004 bd 163.5±10.7 b b: P<0.01 vs. model group; c: P<0.05, d: P<0.01 vs. sham-operated group Compared with the prevention group, the whole-body BMD values were lower in both the prevention and treatment groups (P<0.01), but the lumbar BMD values in the treatment group were still lower than those in the sham-operated group (P<0.01), and the prevention group was also lower than the sham-operated group (P<0.05), suggesting that the indexes in the prevention and treatment groups did not return to normal levels. There was no difference in bone calcium content between the prevention group compared with the treated rats, except for a number of other indexes which tended to be better in the prevention group than the treated group. 2.2 Biomechanical changes As seen in Table 2, the maximum stress, maximum strain and elastic modulus of bone tissue in the sham-operated, prevention and treatment groups were significantly higher than those in the model group, and the differences were statistically significant (P<0.01); compared with the sham-operated group, the maximum stress and maximum strain of bone tissue in the prevention and treatment groups were still lower (P<0.05, P<0.01), suggesting that the indexes in the prevention and treatment groups were not normal levels. Table 2 Comparison of biomechanical changes in the left femur of rats in each group (n=6, ±s) group Maximum stress (mPa) Maximum strain (%) modulus of elasticity (GPa) model group 9.55±1.30 1.002±0.135 0.743±0.073 The sham-operated group 16.27±0.80 b 1.703±0.085 b 1.007±0.093 b Prevention group
14.88±1.03 bc
1.558±0.105 bc 0.948±0.118 b Treatment group 14.03±0.89bd 1.468±0.093 bd 0.955 ± 0.143 b b: P<0.01 compared to model group; c: P<0.05, d: P<0.01 compared to sham-operated group 2.3 Geometric parameters The results are shown in Table 3. The diameters of bone cortical thickness in the sham-operated, preventive and treated groups were significantly higher than those in the model group (P<0.01); the diameters of bone marrow cavities in the preventive and treated groups were lower than those in the model group (P<0.01, P<0.05). Table 3 Comparison of geometrical structural parameters of rats in each group (n=6, ±s) Group Bone cortex thickness (mm) Bone marrow cavity diameter (mm) model group 0.506±0.091 2.120±0.098 sham-operated group 0.755±0.067 b 1.927±0.110 b Preventive group 0.784±0.081 b 1.993±0.149 b Treatment group 0.686±0.077 b 1.975±0.087 a b: P<0.01 compared with the model group, c: P<0.05, d: P<0.01 compared with the sham-operated group 3 Discussion Osteoporosis is defined as a disease in which there is a decrease in systemic bone mass, altered bone tissue microarchitecture, increased bone fragility, and an increased risk of fracture in the absence of trauma or minor trauma. The pathology of this disease is characterized by thinning of the bone cortex, reduced number of trabeculae and smaller volume. The pathophysiological manifestations of osteoporosis induced by removal of ovaries in rats were more similar to those of postmenopausal osteoporosis in humans, with collagen fibers accounting for 90% of the bone organic matter, which is the scaffold for bone salt deposition and the basic structure constituting bone trabeculae. In this experiment, we investigated the effect of the Chinese herbal medicine Gecko extract on the prevention and treatment of osteoporosis by observing the bone mass and biomechanical indexes, aiming to find an efficient, low toxic and safe drug for the prevention and treatment of osteoporosis. The gecko was first published in the Materia Medica, and is listed as the Scutellaria. The gecko is also known as the gecko, the sky dragon, geckos are reptiles, lizards, scorpions, gecko family, animal species for non-webbed geckos or moles, geckos and other species of geckos, currently known geckos are more than 20 species worldwide, mainly distributed in China there are 8 species. In recent years, its dried whole body has been widely used as pills, pills, poultices and other forms of medicine for a variety of clinical diseases, especially for a variety of malignant tumors, tuberculosis, femoral head necrosis, osteomalacia, osteomyelitis, fistulas, sinus tracts and other difficult diseases with definite efficacy, which has attracted more and more widespread attention in the pharmaceutical industry [3]. In this paper, we used the method of decoction and alcoholic sedimentation of dried whole geckos without webbing to prepare gecko extract, and the effect of gavage at a dose of 0.2g per kg body weight was more satisfactory in the pre-experiment, so this dose was chosen as the preventive and therapeutic drug dose. Recently, many advances have been made in the research of Chinese medicine in the prevention and treatment of osteoporosis. For example, the Chinese herb Pueraria lobata can increase estradiol, decrease urinary calcium and urinary deoxypyridinoline, and increase osteocalcin in de-ovulatory rats, indicating that it can increase estradiol, inhibit bone resorption, and promote bone formation [4-5]. The aqueous extract of G. sanguineum significantly increased the bone cortical thickness and the number of osteocytes and bone trabecular area in SAM-P/6 mice [one of the natural aging model mice ( SAM)] [6]. The in vitro osteoblast culture experiments conducted by Han et al. found that: both the serum group of Kudzu Lei and the serum group of Kudzu Lei total flavonoids could directly and significantly promote the proliferation of osteoblasts, increase the protein content and alkaline phosphatase activity of osteoblasts, and reduce the level of Ca content, indicating that Kudzu Lei has a better effect of promoting osteoblast proliferation. enhance the function of osteoblasts [7]. Prednisone caused a significant decrease in bone trabecular area, abnormal bone structure, increased osteoclasts and decreased bone formation rate, accompanied by a decrease in bone inorganic calcium salt and organic strong hydroxyproline, and an increase in blood calcium; Salvia aqueous extract completely counteracted these abnormalities and increased bone dry weight; Salvia aqueous extract also promoted alkaline phosphatase activity in human rat cranial osteoblasts cultured in vitro, in a quantitative and temporal relationship. suggesting that the mechanism of action of Salvia miltiorrhiza against osteoporosis is mainly to promote osteoblast function, promote bone matrix synthesis [8 ]. In this experiment, both preventive and therapeutic administration of Chinese herbal gecko extracts to osteoporotic rats led to significant improvement in the biomechanical indexes of rat bones, which indicated that gecko extracts had the effect of increasing bone strength; and the trend that several test indexes of bone density values in the preventive group were better than those in the therapeutic group indicated that the preventive medicine could strengthen the compensatory function of the body and prevent the occurrence of osteoporosis, and the above results were consistent with Similar effects of single herbal medicine were reported in the literature. In addition, the bone density, bone calcium content and bone cortical thickness of the rats in the preventive and treatment groups were significantly higher than those in the model group, and the increase in bone density of the lumbar spine was higher than that of the whole body, suggesting that Gecko extract has the effect of increasing bone mass, and its effect on cancellous bone is better than that on cortical bone. In conclusion, the gecko extract had some preventive and therapeutic effects on osteoporosis rats. We believe that the possible mechanism is that the gecko extract contains a variety of amino acids and other active substances, which mainly affect the bone metabolism such as the increase of collagen fiber formation, thus improving the bone strength and bone mass, the mechanism of which needs further study.