冬虫夏草及其单体组分对大鼠骨质疏松治疗作用及机制的实验研究

发布时间：2018-07-16 13:46

【摘要】：骨质疏松症（Osteoporosis，OP）是多种原因引起的一种以骨组织含量减少，骨的微观结构退化为特征，致使骨的脆性增加、强度降低而导致易于发生骨折的一种全身性骨骼疾病。骨质疏松症已经不仅仅是一种疾病，目前已成为一个严重的社会问题。它带来的最主要症状有剧烈的疼痛、多发骨折以及胸腔缩小引起的呼吸困难。在众多承受骨质疏松症病痛折磨的患者中，绝经后女性因受到衰老和体内激素水平下降的双重影响，成为了该病最大的受害人群，约占总患病人数70%以上。另外，随着糖尿病患者日渐增多，特别是中老年人居多，因糖尿病引起的骨质疏松症也成为目前临床上骨质疏松症形成的重要原因之一，并且由于其具有隐匿性而往往被患者忽视，延误治疗，，其后果不堪设想。目前防治骨质疏松症的药物主要是骨吸收抑制剂、骨形成促进剂和矿化药物，这些药物均属替代治疗，在长期使用过程中，这些药物具有毒副作用大，靶向性低，依从性差等缺点。因此，有必要找寻到一种能够具有广泛受众人群的药物来治疗骨质疏松症。中药的应用在我国已有几千年的历史，在人们心中具有极高的认同度。中药在治疗骨质疏松症方面具有全身调理、毒副作用小、取材方便等优势，越来越受到人们的关注。而在众多中药中，冬虫夏草特有的药用价值长久以来一直受到人们的青睐。因此，如何发掘出冬虫夏草的潜质，研发出具有自主知识产权的抗骨质疏松中药，成为了我们研究的目的所在。那么，冬虫夏草是否具有预防和治疗骨质疏松症的能力呢？它发挥拮抗骨质疏松作用的机制是什么？作为冬虫夏草重要单体成分的虫草多肽是否具有抗骨质疏松的潜力呢？我们将围绕着上述疑问展开研究。 1.富含锶的冬虫夏草对去卵巢骨质疏松大鼠的作用及机制研究（1）目的：研究富含锶的冬虫夏草对去卵巢大鼠骨质疏松的治疗作用和相关机制。（2）方法：将雌性大鼠随机分为5组：假手术组（Sham）、去势组（OVX）、冬虫夏草组（CS）、锶组（SR）以及富含锶的虫草组（CSS）。每组10只。4组通过手术摘除卵巢(OVX)，另一组进行假手术（对照）。第1组（假手术）和第2组(OVX)经口接受10ml生理盐水治疗，第3组、第4组和第5组分别经口接受10ml CSS、CS和SR治疗，时间长达8周。观察大鼠尿钙、血钙、血磷、骨矿物质密度（BMD）、骨矿物质含量（BMC）以及子宫、胸腺和体重的变化情况。测定各组大鼠血清碱性磷酸酶(ALP)、抗酒石酸酸性磷酸酶(TRAP)、血清骨钙素(OC)、同型胱氨酸（HCY）、I型胶原交联型羧基端肽(CTX)、雌二醇和干扰素-γ (IFN-γ)水平。（3）结果：CSS和SR均对去卵巢骨质疏松大鼠的骨质机械强度和矿物质含量产生了积极的影响。但是，CSS治疗组的股骨颈强度高于SR治疗组。CSS和SR可显著降低尿钙、血钙以及血磷水平。相反， CS和CSS可显著增加萎缩子宫的重量和体重，并且还减轻动物胸腺质量，而SR没有表现出任何此类作用。同时CSS可以显著降低血清ALP和TRAP水平并促进骨钙素的分泌并降低CTX水平。另外，CSS可以明显降低IFN-γ水平并显著提高体内雌二醇的水平。（4）结论：本研究证明了富含锶的冬虫夏草在治疗人类绝经后骨质疏松症中的价值。CSS可以有效治疗因骨强度和骨矿含量下降引起的骨质疏松症，其作用机制主要是通过减少骨质疏松模型中骨质的流失，显著减少骨吸收，促进骨生成并促进体内雌二醇生成实现的。同时也证明了冬虫夏草同样具有拮抗骨质疏松症的潜力。 2.冬虫夏草提取物对大鼠废用性骨质疏松的预防作用（1）目的：验证冬虫夏草是否具有预防大鼠废用性骨质疏松症的作用。（2）方法：将大鼠随机分为6组，其中5组做后肢悬吊（HLS）处理。其中1个HLS组经口给予阿仑膦酸钠（2.0mg/kg/天），另外3个HLS组分别经口给予不同剂量的冬虫夏草（100、300、500mg/kg/天），连续8周，余下的HLS组作为HLS之前和之后不给药的对照组。每组分别由10只雄性和雌性组成。对体重、血清和尿液中的生化指标、骨矿物质密度（BMD）、骨矿物质含量（BMC）、机械力学和骨微结构进行测定。（3）结果：与其它HLS组相比，使用较高剂量（300和500mg/kg/天）冬虫夏草或阿仑膦酸钠的治疗组在体重、机械强度、BMD和BMC等方面产生了积极作用。在HLS大鼠中，冬虫夏草呈剂量依赖性地使骨转换标志物下降，并使骨钙素水平升高。L-4椎体的显微CT分析结果表明，冬虫夏草高剂量组（500mg/kg）大鼠的骨小梁无论是数目、厚度还是骨小梁的间隙都比HLS组得到了明显的改善。（4）结论：本研究表明，经过8周使用较高剂量的冬虫夏草可以预防大鼠废用性骨质疏松症。意味着冬虫夏草也可以作为预防人类废用性骨质疏松症的替代疗法。 3.虫草多肽对四氧嘧啶诱发的糖尿病大鼠骨质疏松的作用研究（1）目的：研究虫草多肽对四氧嘧啶诱发的糖尿病大鼠骨质疏松症的保护作用及可能的机制。（2）方法：将大鼠随机分为5组，其中4组给予腹腔注射四氧嘧啶构建糖尿病模型。3组糖尿病大鼠每天接受腹膜内注射用虫草多肽（Cordymin）（20，50，和100mg/Kg/天），共5周。余下的糖尿病组对照组。每组分别由10只雄性大鼠组成。对血糖、糖化血红蛋白（HbA1c）、血清碱性磷酸酶（ALP）、抗酒石酸酸性磷酸酶（TRAP）、胰岛素水平、总体抗氧化剂活性（TAOS）、骨矿物质密度（BMD）、骨矿物质含量（BMC）以及胰岛β细胞损伤情况进行测定和评估。（3）结果：虫草多肽可以降低血糖、糖化血红蛋白（HbA1c）、血液中ALP及TRAP水平，并呈剂量依赖性的升高胰岛素水平。此外，糖尿病大鼠经过虫草多肽的治疗可以相对减少胰腺β细胞的死亡并降低糖尿病大鼠总体抗氧化剂活性。同时可以增加糖尿病大鼠的BMC和BMD。（4）结论：虫草多肽对糖尿病引起的骨质疏松症可能具有一定的保护作用。综上所述：以上研究结果说明：冬虫夏草能够促进成骨、降低骨质流失，并且可以有效的修复了骨质疏松对机体机能的损害。而作为冬虫夏草中重要的单体成分之一的虫草多肽可以有效缓解糖尿病症状，减轻胰腺β细胞损伤，有效拮抗体内氧化剂的活性，增加体重，促进体内骨钙的沉积，甚至具有治疗因糖尿病引起的骨质疏松症的潜力。这一重大的发现为我们找寻到了治疗骨质疏松症的新的方向，有望拓展对冬虫夏草药理学功能和抗骨质疏松作用的新认识，为今后研发具有自主知识产权的抗骨质疏松药物奠定坚实的理论基础。
[Abstract]:Osteoporosis (OP) is a kind of systemic bone disease caused by the decrease of bone tissue and the degeneration of bone microstructure, resulting in the increase of bone brittleness and the decrease of strength, which leads to the fracture of a systemic bone disease. Osteoporosis is not only a disease, but has become a serious society. The main symptoms are severe pain, multiple fractures and dyspnea caused by narrowing of the chest. Among the many patients suffering from osteoporosis, postmenopausal women have become the largest victims of the disease, accounting for about 70% of the total number of diseases due to the double effects of aging and decreased hormone levels in the body. On the other hand, with the increasing number of diabetic patients, especially in the middle and old people, the osteoporosis caused by diabetes is also one of the important reasons for the formation of osteoporosis in clinical. And because of its concealment, it is often ignored by the patients, and the result is unimaginable. The medicine for the prevention and treatment of osteoporosis is at present. Substances are mainly bone resorption inhibitors, bone formation accelerators and mineralized drugs. These drugs are all alternative treatments. During the long term use, these drugs have the disadvantages of large toxic and side effects, low targeting and poor compliance. Therefore, it is necessary to find a drug that can be widely used in the treatment of osteoporosis.
The application of traditional Chinese medicine has a history of thousands of years in our country and has a high degree of recognition in people's heart. Traditional Chinese medicine has the advantages of whole body conditioning, small toxic and side effects and convenient selection of materials in the treatment of osteoporosis, and more and more people pay attention to it. In many Chinese medicine, the special medicinal value of winter insect and summer grass has been subject to people for a long time. Therefore, how to discover the latent quality of the Cordyceps sinensis and develop the anti osteoporosis Chinese medicine with independent intellectual property rights has become the purpose of our research. Then, is the ability of Cordyceps sinensis to prevent and treat osteoporosis? What is the mechanism of antagonizing osteoporosis? As important for Cordyceps sinensis Whether the single component of Cordyceps polypeptide has the potential to combat osteoporosis? We will focus on these questions.
Effect and mechanism of 1. strontium rich Cordyceps sinensis on ovariectomized rats
(1) objective: To study the effect and mechanism of Cordyceps sinensis (SR) on osteoporosis in ovariectomized rats.
(2) method: the female rats were randomly divided into 5 groups: the sham operation group (Sham), the castration group (OVX), the Cordyceps sinensis group (CS), the strontium group (SR) and the strontium rich Cordyceps group (CSS). 10 groups of.4 groups in each group were operated on the ovaries (OVX), the other group was sham operation, and the first groups (sham operation) and the second group (OVX) received 10ml physiological saline treatment and third, Fourth groups and fifth groups were treated with 10ml CSS, CS and SR for up to 8 weeks. The changes of urinary calcium, blood calcium, blood phosphorus, bone mineral density (BMD), bone mineral content (BMC), uterus, thymus and body weight were observed. Serum alkaline phosphatase (ALP), tartaric acid phosphatase (TRAP), and Serum Osteocalcin (OC) were measured in each group. Homocysteine (HCY), type I collagen cross-linked carboxy terminal peptide (CTX), estradiol and interferon gamma (IFN- gamma) level.
(3) results: both CSS and SR had a positive effect on the bone mechanical strength and mineral content in ovariectomized rats. However, the femoral neck strength of the CSS treatment group was higher than that of the SR treatment group,.CSS and SR significantly decreased the urinary calcium, blood calcium and blood phosphorus levels. On the contrary, CS and CSS significantly increased the weight and weight of the atrophied uterus, and also had a significant increase in the weight and weight of the atrophied uterus. The quality of the thymus was reduced, and SR did not show any such effect. At the same time, CSS could significantly reduce the level of serum ALP and TRAP, promote the secretion of osteocalcin and reduce the level of CTX. In addition, CSS can significantly reduce the level of IFN- gamma and significantly increase the level of estradiol in the body.
(4) conclusion: This study proves that the value of strontium rich Cordyceps sinensis in the treatment of postmenopausal osteoporosis can effectively treat osteoporosis caused by the decrease of bone strength and bone mineral content. The main mechanism is to reduce bone loss, reduce bone absorption and promote bone formation by reducing bone loss in the osteoporosis model. It also promotes the production of estradiol in vivo. It also proves that Cordyceps has the potential to combat osteoporosis.
Preventive effect of 2. Cordyceps sinensis extract on disused osteoporosis in rats
(1) objective: to test whether Cordyceps sinensis has the preventive effect of disused osteoporosis in rats.
(2) method: the rats were randomly divided into 6 groups, of which 5 groups were treated with rear limb suspension (HLS), of which 1 groups were given alendronate (2.0mg/kg/ days) through the mouth, and the other 3 HLS groups were given different doses of Cordyceps (100300500mg/kg/ days) for 8 weeks, and the remaining HLS group was used as the control group before and after HLS. Do not consist of 10 males and females. Biochemical markers in body weight, serum and urine, bone mineral density (BMD), bone mineral content (BMC), mechanical mechanics, and bone microstructures were measured.
(3) results: compared with other HLS groups, the treatment group using Cordyceps sinensis or alendronate with higher doses (300 and 500mg/kg/ days) had a positive effect on body weight, mechanical strength, BMD and BMC. In HLS rats, Cordyceps decreased dose-dependent bone conversion markers and increased the level of osteocalcin in the.L-4 vertebral body. The results of micro CT analysis showed that the bone trabecula in the high dose group of Cordyceps sinensis (500mg/kg) rats, whether the number, the thickness or the bone trabecular space, had been significantly improved than that of the HLS group.
(4) conclusion: This study shows that the use of Cordyceps sinensis after 8 weeks of high dose of Cordyceps can prevent disuse osteoporosis in rats, which means that Cordyceps sinensis can also be used as a substitute for the prevention of human waste.
Effect of 3. Cordyceps polypeptide on osteoporosis induced by alloxan in diabetic rats: a study of four
(1) objective: To study the protective effect of Cordyceps polypeptide on osteoporosis induced by alloxan in diabetic rats and its possible mechanism.
(2) method: the rats were randomly divided into 5 groups, of which 4 groups were given intraperitoneal injection of four oxouracil to construct diabetes model.3, the diabetic rats were treated by intraperitoneal injection of Cordymin (20,50, and 100mg/Kg/ days) for 5 weeks. The remaining diabetic control group. Each group was composed of 10 male rats respectively. White (HbA1c), serum alkaline phosphatase (ALP), anti tartaric acid acid phosphatase (TRAP), insulin level, total antioxidant activity (TAOS), bone mineral density (BMD), bone mineral content (BMC), and islet beta cell damage were measured and evaluated.
(3) results: Cordyceps can reduce blood sugar, glycated hemoglobin (HbA1c), ALP and TRAP levels in the blood, and increase the level of insulin in a dose dependent manner. In addition, diabetic rats can reduce the death of pancreatic beta cells and lower the overall antioxidant activity of diabetic rats by the treatment of Cordyceps polypeptide. BMC and BMD. in diabetic rats
(4) conclusion: Cordyceps polypeptide may have some protective effects on osteoporosis caused by diabetes.
In summary, the above results show that Cordyceps sinensis can promote osteogenesis, reduce bone loss, and can effectively repair the damage to the body function of osteoporosis. As one of the important monomers in the Cordyceps sinensis, the Cordyceps can effectively alleviate the symptoms of diabetes, reduce the pancreatic beta cell damage, and effectively antagonize it. The activity of the oxidant in the body increases the body weight, promotes the deposition of bone calcium in the body, and even has the potential for the treatment of osteoporosis caused by diabetes. This major discovery provides us with a new direction for the treatment of osteoporosis and is expected to expand the new understanding of pharmacological function and anti osteoporosis effect of Cordyceps sinensis for the future. The research and development of anti osteoporosis drugs with independent intellectual property rights will lay a solid theoretical foundation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R285.5

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