Studies On The Mechanism Of E Jiao Bu Shen Jian Gu Formula In Model Rats With Osteoporosis

Objective:The aim of this study is to investigate the osteoporosis from atheoretical point of view with different traditional Chinese and westernmedical methods，to observe the usual manifestations of rats whichwere completely removal of ligation and ovariectomy，the changes ofbone pathological feature，bone mineral density，bone biomechanics，blood biochemistry and the expression of25（OH）D₃、1,25（OH）₂D₃withthe osteoporotic animal models through experimental study，to explorethe effects and mechanism of treating osteoporosis and provide ascientific basis for treating osteo-porosis with E Jiao Bu Shen Jian GuFormula.Methods:Seventy female SD rats aged8months， postmenopausalosteoporotic animal model was established. One week after the feedingadaption，rats were randomly divided into four groups: sham-operatedgroup and model group，17in each group.36rats were randomlydivided into group A of osteoporotic animal model with Alendronatesodium and group E of osteoporotic animal model with E Jiao Bu ShenJian Gu Formula，18in each group. Rats in sham-operated group were fed as usual；rats in model group were intragastrically administratednormal physiological saline for90days，rats in other groups wereintragastrically administrated for90days. All rats were stopped treatingfor a day for containing blood.To observe the usual manifestations and the changes of weightfrom living rats.After treated for3months，2％pentobarbital sodiumintraperitoneal anesthesia，getting blood from the arteria femoralis forreserve and writing down the weight of both kidneys，offside femur anduterus after three months. Soft tissue was removed completely from therats’ offside femur. To use of LUNAR-Prodigy X-ray absorptiometry ofoffside femur and the bone mineral density of bothends of femurmeasured in rats.Then biomechanical detector to determine themaximum load （ML） and stiffness. Femur of left side was putted andfixed in neutral Formaldehyde solution. And then conventional HEstaining，making pathological section as required，observing thechanges of bone tissue under the Microscope. The serumosteocalcin（OCN），the procollagen I carboxyterminal propeptide（PICP），25-hydroxyvitamin D₃，[25（OH）D₃]，1,25-dihydroxyvitaminD₃[1,25（OH）₂D₃] were detected by enzyme-linked immunosorbentassay （ELISA）. The expression of Vitamin D receptor（VDR） in kidneytissue was detected by Polymerase Chain Reaction（PCR） method.Statistical analysis: SPSS15.0statistical software were used foranalysis of variance，all data were represented by mean（x±s）standarddeviation，P values of <0.05were considered statistically significant.Results:（1）The usual manifestations of ratsCompared with sham-operated rats，spirits of in model group weresagged、furious anxiety and were slow in reacting；Their hair was not brightness，untidy；Some rats were less physically active and somnolent.Compared with group A and group E，rats in the two groups wereemotional stability，in good spirits，lively，responsive and their hair wassupple，brightness during treatment.（2）Changes of weight of ratsCompared with sham-operated，weight of rats in model group wasapparently heavier than sham-operated rats after two months. Weightof rats in model group E was gradual increase，which was similar to thesham-operated rats.There was statistically significant difference.However，rats in group A gain weight obviously.（3）Index of uterus of ratsCompared with sham-operated group，index of uterus wassignificantly decreased in other groups（P＜0.05）；Index of uterus ofgroup E was superior to that of the group A.There was obviousdifferences in statistical significance.（4）Index of both kidneys of ratsIndex of both kidneys of model group was significantly lower thansham-operated group （P＜0.05）； Index of both kidneys of group Eand group A was significantly higher than model group. Index of bothkidneys of group E was superior to that of the group A. There wasobvious differences in statistical significance.（5）Weight of femur of ratsCompared with sham-operated group，weights of femur weresignificantly decreased in other groups（P＜0.05）； Weights of femur ofgroup E and group A were higher than model group. And the weight offemur of group E was superior to group A. There was obviousdifferences in statistical significance.（6）Observation of pathological section of bone tissue Sham-operated rats femur bone were rich，continuous，dense，however，rats in model group were sparse trabeculae，irregular；Compared with model group，there was no obvious decrease intrabeculae bone，arranged regularly，continuous and intact. Comparedwith model group，bstructural changes of bone morphology wassignificantly difference in group E and group A.（7）Differences of BMD of ratsBMD of femur of model group was significantly lower thansham-operated group. From statistical analysis，there was obviousdifferences in statistical significance（P＜0.05）. After treated for3months，compared with model group，bone mineral density of femur ofother groups was significantly increased（P＜0.05）.Compared group E and group A，the changes of bone mineral densitywas unconspicuous，the scope was similar. There was no significantlydifference between group E and group A in BMD of femur.（8）Results of Biomechanics of ratsCompared with sham-operated group，the largest compressivestress and stiffness were obvious decreased in femur of rats in modelgroup，P＜0.05；compared with model group，the largest compressivestress and stiffness were significantly increased in femur of rats ingroup E and group A. And the largest compressive stress and stiffnessof group E was superior to group A. There was statistically significantdifference.（9）Changes of OCN and PICP of ratsOCN and PICP were lower than sham-operated group，P＜0.05；and OCN and PICP of group E and group A were higher than modelgroup P＜0.05. Effect of OCN of group A was a little higher than groupE；however，effect of PICP of group E was superior to group A. There was statistically significant difference.（10）Gene level of25（OH）D₃、1,25（OH）₂D₃of ratsConcentration of25（OH）D₃，1,25（OH）₂D₃o in model group wasobvious lower than sham-operated group （P＜0.05）；however theconcentration of25（OH）D₃，1,25（OH）₂D₃in group E and group A washigher than model group （P＜0.05）. And group E was superior to groupA. There was statistically significant difference.（11）Expression of VDR-mRNA in kidneys tissueCompared with sham-operated group，expression of VDR-mRNA inkidneys tissue of model group were statistically significant difference.After treated for3months，expression of VDR-mRNA on gene level inkidneys tissue of groups were higher than model group （P＜0.05）；Andexpression of VDR-mRNA on gene level in kidneys tissue of group Ewas superior to group A. There was statistically significant difference.Conclusion:（1）There is significantly difference between sham-operated groupand ovariectomized rats，as a result，to build a animal model ofosteoporosis to meet the requirements of this experiment successfully.（2）The result shows that E Jiao Bu Shen Jian Gu Formula canimprove general performance of ovariectomized rats. Its effects whichadjust lipid metabolism and prevent gain weight rapidly are similar toEstrogen. E Jiao Bu Shen Jian Gu Formula can not only increase indexof uterus，both kidneys and weight of bone but also put off a loss ofbone mass，increase BMD and improve bone ultrastructure，strength ofbone. E Jiao Bu Shen Jian Gu Formula can not only improve theconcentration of OCN，PICP，25（OH）D₃and1,25（OH）₂D₃but alsoincreasing regulate the VDR-mRNA. There was a better prevention andtreatment that E Jiao Bu Shen Jian Gu Formula can be treated for osteoporosis. The mechanism may be multi-channel，multi-target andmulti-step not only to promote metabolism of calcium and phosphorus inbone， bone formation and bony remodeling but also to controlosteoclast proliferation，osteocyte and osteoblast apoptosis.