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Biol Trace Elem ResDOI 10.1007/s12011-010-8711-4 The Co-effect of Cordyceps sinensis and Strontiumon Osteoporosis in Ovariectomized Osteopenic Rats Wei Qi & Ya-bo Yan & Pu-jie Wang & Wei Lei Received: 14 April 2010 / Accepted: 21 April 2010 # Springer Science+Business Media, LLC 2010 Abstract The co-effect of Cordyceps sinensi (CS; caterpillar fungus) and strontium onovariectomized osteopenic rats was studied in this paper. After the rats were treated orallywith CS, strontium (SR), and CS rich in strontium (CSS), respectively, the urine calcium,plasma calcium, plasma phosphorus, bone mineral content, mechanical testing, and themass of uterus, thymus, and body were examined. Both CSS and SR have a positive effecton mechanical strength and mineral content of ovariectomized osteopenic rats. However,femoral neck strength in the CSS-treated group was higher than those in the SR-treatedgroups. CSS and SR significantly decreased urinary calcium excretion and plasma totalcalcium and inorganic phosphate concentrations. On the contrary, CS and CSS significantlyincreased weights of atrophic uteri and weights of body and also decreased the thymusmass in animals, whereas SR did not exhibit any such effects. Our experiments havedemonstrated that CSS possess a preferable effect against the decrease of bone strength andbone mineral mass caused by osteoporosis. It was caused by the co-effect of CS andstrontium. The mechanism of it includes decreases bone resorption, increases boneformation, increases in body weight, and enhances 17β-estradiol-producing as well asenhancing the immune functions in animals. The data provide an important proof ofconcept that CSS might be a new potential therapy for the management of postmenopausalosteoporosis in humans.
Keywords Strontium . Cordyceps sinensis (caterpillar fungus) . Osteoporosis . Calcium .
Inorganic phosphate W. Qi Y.-b. Yan W. Lei (*)Department of Orthopaedics, Xi Jing hospital, The Fourth Military Medical University, Xi’an 710032,People’s Republic of Chinae-mail: leiwei@fmmu.edu.cn W. Qi P.-j. WangThe Surgery Department of 520th Hospital of PLA, Mian yang 621000, People’s Republic of China Strontium (Sr) is a member of the alkaline earth elements and occupies an intermediateposition between calcium and barium. The currently available data indicate that strontiumhas metabolic effects on bone in vivo. There is experimental evidence that strontiumadministration at low dose reduces bone resorption and increases bone formation, resultingin increased bone mass in normal animals []. By virtue of these biologic effects,strontium compounds are potentially useful treatments for a variety of diseases that arecharacterized by accelerated bone loss. It has been shown to reduce the risk of osteoporoticfractures in two large phase III clinical trials (the Spinal Osteoporosis TherapeuticIntervention study [SOTI] and the Treatment Of Peripheral OSteoporosis Study [TROPOS])[However, the toxicity associated with strontium limits its role as a therapeutic agentfor osteoporosis. Typical clinical manifestations are diarrhea, vomiting, and venousthromboembolism (VTE) including pulmonary embolism []. To reduce the toxicity ofstrontium, the co-effect of Cordyceps sinensis (CS) and strontium on osteoporosis wasstudied in this paper.
CS (caterpillar fungus) is a fungus and has been known as a traditional medicine in China. Many studies have shown that CS possesses antifatigue ], antitumor [antioxidant [], immunomodulatory [hypoglycemic [], and vasorelaxantactivities ]. In addition, it modulated steroidogenesis. In the postmenopausal years,estrogen deficiency leads to excessive bone resorption, bone loss, and eventuallyosteoporosis [Rat adrenal cells treated with CS show increased corticosteroneproduction, and this effect is mediated through protein kinase C ].
Another important property of fungus is the ability to take up and accumulate trace metals such as cadmium, lead, arsenic, copper, nickel, silver, chromium, and mercury in thebody or mycelium of the fungus The purpose of this study was to investigate theeffect of CS rich in strontium (CSS) on osteoporosis.
Strontium ranelate was purchased from Betapharma Co., Ltd., Shanghai, China.
The seed of CS was purchased from the Agricultural Culture Collection of China. Five tosix pieces of the mycelia of CS were transferred from a slant into 500-mL Erlenmeyerflasks containing 300 mL of fermented culture medium (20% potato extract liquid +2.0%dextrose +0.1% KH2PO4+0.05% MgSO4+0.9% C12H6N2O8SSr8). The culture wasincubated at 27°C on a rotary shaker at 180 rmp for 5 days. The 120-hour-old fermentedliquid culture was CSS. An ampule was filled with 10 mL of CSS stirred by a homogenizerand then was sterilized in microwave oven for 3 minutes.
The fermented mushroom of CS was produced using the same method to produce CSSexcept that there was no C12H6N2O8SSr8 in the fermented culture medium.
The Co-effect of Cordyceps sinensis and Strontium on Osteoporosis in Ovariectomized Osteopenic Rats Strontium ranelate (0.9 g) was dissolved in 100 mL of normal saline. An ampule was filledwith 10 mL of SR and then was sterilized in a microwave oven for 3 minutes.
Fifty female Wistar rats (2 months old and weighing 225±25 g) were used in the study.
This study was performed in accordance with the Guide for the Care and Use of LaboratoryAnimals. Care was taken to minimize discomfort, distress, and pain to the animals.
The rats were randomly divided into five groups of animals, four ovariectomized (OVX) and another was given a sham-operation (control). Groups 1 (sham) and 2 (OVX) were treatedorally with 10-mL of saline; groups 3, 4, and 5 were treated orally with 10-mL of CS, SR, andCSS for 8 weeks, respectively. Body weight of the animals was recorded weekly.
On the last day of treatment, urine was collected by micturation induced by manual pressure from overnight fasted animals and preserved at -20°C until further analysis [At necropsy, blood was collected from the dorsal aorta under ether anesthesia. Aftercentrifugation, serum was harvested and kept at -20°C until analysis. Uteri were isolated.
The absolute weight of uterine tissue was recorded and normalized with body weight(relative weight of uterus, i.e., weight of uterus per 100 g of body weight) of animals. Themasses of thymus were also determined. The femoral neck was processed for mechanicaltesting. The left femur and L-4 vertebra bone were processed for mineral content.
Calcium content of urine was measured by flame photometry (EHSY, China) after suitabledilution with double-distilled water Serum samples were analyzed for their calcium and phosphorus contents using thearsenazo-3 dye and ammonium molybdate colorimetric methods, respectively.
The left femur and L-4 vertebra were mineralized at the temperature of 620°C for 48 hoursand weighed. The mineralized bones were dissolved in 6 M HCl, and then calcium contentin the bone mineral content was assayed using a colorimetric method.
The mechanical strength of the femoral neck was measured with the method described byMa and Fu Briefly, soft tissues were removed from the femora and the shafts of thefemora cut at the midshaft of each femur. The distal femora were fixed withmethylmethacrylate cement up to the lesser trochanter, maintaining a vertical position. Avertical load from a brass cylinder was applied to the top of the femoral head. The cylinderwas directed parallel to the axis of the femoral diaphysis and moved at a constantdisplacement speed of 5 mm/min until the femoral neck fractured. The fracture load wasrecorded at the peak force as Newton (N) at the point that the femoral neck fractured.
Group means were compared by ANOVA with GraphPad Prism (GraphPad Software, Inc.).
Multiple comparison tests were performed with Tukey for significant differences at p<0.05.
Ovariectomized (OVX) animal models, in a variety of species, have been used to evaluatethe mechanism of or to assess the effect of drugs in estrogen deficiency. Strontiumcompounds are potentially useful treatments for a variety of diseases that are characterizedby accelerated bone loss. CS could enhance the fecundity of women by modulating the17β-estradiol-producing capacity of granulosa cells [So the co-effect of CS andstrontium on osteoporosis was studied in this paper.
The body weights of the rats are presented in Fig. . Contrasted with the other groups, thebody weights of rats in the CSS- and CS-treated groups increased gradually 5 weeks later(p<0.05 and p<0.01). On the contrary, the body weights of rats in the SR-treated group didnot increase significantly (p>0.05). It indicates that CS could supplement nutrients to rats.
According to ancient descriptions, CS possesses important pharmacological activities forprotecting lung and kidney functions and in nourishing essential and vital energy Body weight is a general index of the health of an animal. The increases in body weight byCSS may contribute to maintaining the elevated periosteal bone formation and inhibitendosteal bone resorption ].
Absolute and relative uterine weight was significantly lower in OVX rats than in sham rats. Significant increase was observed with CS and CSS (p<0.05), whereas SR did notshow any increase in weight of atrophic uterus (Table ). Uterus is a primary target organfor estrogen. The increases in uterus weight by CSS may account for the possiblemechanism of action.
The ovariectomized rats were shown by substantial increases in the thymus mass in relation to the sham-operated control rats (Table CS and CSS significantly decreased thethymus mass of the ovariectomized rats (p<0.01), whereas SR did not show any decrease in Fig. 1 Body weight changes for the study. (◆ sham group, □ OVX group, ■ SR group,▲ CSS group, and ■CS group) CSS and CS significantly increased body weights compared to OVX animals, whereas the bodyweights of rats in the SR group did not increase significantly (p>0.05). Values are mean ± SEM, n=10. *p<0.05, **p<0.01 compared to the OVX group at the same time point The Co-effect of Cordyceps sinensis and Strontium on Osteoporosis in Ovariectomized Osteopenic Rats Table 1 Effects of CSS, CS, and SR on the Mass of Uterus and Thymus in Ovariectomized Rats Relative weight of uterus is the weight of uterus normalized with body weight (mg per 100 g of bodyweight). Values are mean ± SEM, n=10* p<0.05 vs. OVX control; ** p<0.01 vs. OVX control weight of thymus. The hypertrophy of the thymus resulting from ovariectomy was inhibitedby CS and CSS. Recent scientific studies have shown that CS is capable of modulatingimmune responses. The results indicate that CS and CSS could enhance the immunefunctions in OVX animals. On the contrary, SR exhibited undesirable side effects, such as aslower rate of weight gain in uterus and body.
The effects of CSS were investigated in OVX rats by calcium balance and calcium kineticstudies. In the present study, significant increase in urinary Ca excretion was observed in OVXcontrol. Although CS decreased urinary Ca excretion, it was not significant (Table On thecontrary, CSS and SR significantly decreased urinary Ca excretion (p<0.01 and p<0.05).
It has been reported that the serum biochemical parameters, such as Ca and P, are normal inosteoporosis in man ]. However, in the present investigation, significant differences inCa and P levels were found in different group. The effects of treatment with CSS, SR, andCS on plasma total calcium and inorganic phosphate concentrations are shown in Table .
All of the treatments significantly decreased plasma total calcium and inorganic phosphateconcentrations. However, the values of the CS-treated group (12.87 and 8.51 mg/dL,respectively) were significantly higher than those of the CSS- and SR-treated group.
Table 2 Effect of CSS, CS and SR on Serum Calcium (Ca), Inorganic Phosphorus (P), and Urinary CalciumExcretion in Ovariectomized Rats Values are mean ± standard deviation, n=10* p<0.05 vs. OVX control; ** p<0.01 vs. OVX control Table 3 Bone Mineral Content Measurement (Mean ± Standard Deviation, n=10) The different letters in the same column indicate a statistical difference (p<0.05) The administration of CSS to the ovariectomized animals increased significantly themineral content in the examined bones when compared to the ovariectomized group(Table ). However, the same results did not occur in the CS- and SR-treated groups. Itagrees with the above results of the increases in body weight by CSS. It is consistent withthe finding that body weight may contribute to maintaining the elevated periosteal boneformation and inhibit endosteal bone resorption [ Mechanical strength of bones is the most important parameter related to fracture risk. In thepresent study, we have analyzed mechanical strength parameters that represent extrinsicproperties of the bone []. The average maximum fracture loading to the femoral neckswas 30.2 and 28.3% lower in the CS and SR groups compared with the CSS group,respectively (Fig. ). It is worth noticing that femoral neck strength in the CSS-treatedOVX group was higher than that in the SR-treated OVX group.
The results implied that therapeutic effect on ovariectomized osteopenic rats was caused by the co-effect of CS and strontium. The mechanism of CSS on ovariectomized osteopenic ratsincludes decreases bone resorption, increases bone formation, increases in body weight, andenhances 17β-estradiol-producing as well as enhances the immune functions in OVX animals.
Fig. 2 Mechanical strength of the femoral neck. The data are presented as mean ± SD (n=10 per group).
*p<0.05, **p<0.01 compared to the OVX group The Co-effect of Cordyceps sinensis and Strontium on Osteoporosis in Ovariectomized Osteopenic Rats This study clearly shows that CSS is able to counteract the bone loss in an experimentalmodel of established osteoporosis. These findings suggested that the potency of CSS is dueto effects on decreases bone resorption, increases bone formation, increases in body weight,and enhances 17β-estradiol-producing as well as enhances the immune functions in OVXanimals. The results suggest that CSS possess a preferable effect against the decrease ofbone strength and bone mineral mass caused by osteoporosis. The data provide animportant proof of concept that CSS might be a new potential therapy for the managementof postmenopausal osteoporosis in humans.
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