Experimental Study On The Relationship Between Activity Of Hepatic CYP3A And Steroid-Induced Femoral Head Osteonecrosis And Effect Of The Jiangufang To The Hepatic CYP3A Activity In A Rabbit Model

ObjectiveOsteonecrosis of the femoral head (ONFH) is regard as one of the difficult to treat disease in the field of Orthopedics. ONFH can be divided into traumatic and non-traumatic causes. ONFH which is caused by the traumatic mainly because of injury of hip such as neck fracture and hip dislocation, etc.. The non-traumatic ONFH is caused by a long-term corticosteroids use and Alcoholism. A long-term corticosteroids use is the most causes ONFH in the non-traumatic part, it is about 50%. ONFH is a progressive disease, without effective treatment and prevention, about 80% people pathogenesis femoral head collapse in 1 to 4 years, the event of a femoral head collapse will develop into a serious arthritis in the majority of patients, finally the arthroplasty is needed. non-traumatic ONFH is regularity occurs in middle-aged, middle-aged in the long term effect of arthroplasty surgery is still difficult to predict. Therefore, the search for effective treatment of hip protection is especially important, early diagnosis and early intervention is the premise to have achieved excellent therapeutic effect. Explore the ONFH pathogenesis, find the sensitive methods for early diagnosis and effective early treatment, it has has very important significance. Clinical data indicate that the ONFH caused by glucocorticoids is systemic disease but performance in the part of the body. Advantages of Chinese medicine treatment of systemic drug therapy to the whole to reduce the side effects of hormone application, prevention the high-risk groups, sub-clinical phase and before the collapse to use individual differentiation therapy. As the complexity of disease pathogenesis and genetic background characteristics, it was quite difficult to determine high-risk groups accurately. Because of lack of clinical symptoms and X-ray performance within the sub-clinical phase and before the collapse, patients often less liked to notice even not to adopt sensitive but expensive magnetic resonance imaging (MRI) performance. As the early diagnosis was difficult, it couldn't use Chinese medicine to prevention effectively and treat before the collapse. Therefore, it was necessary to research the ONFH caused by glucocorticoids in-depth.Clinical study found that the use of high-dose hormone therapy is significant risk factor for ONFH. But the same hormone dosage in different patients are often comes out different results, some patients will lead to ONFH, some patients not. that means the individual to hormones is different of sensitivity. The metabolism of glucocorticoids in the body is the key enzyme cytochrome P4503A enzymes (CYP3A), researchers found that the activity of CYP3A large range of low activity of CYP3A, may cause the serum clearance rate of corticosteroids due to the decreasing level of free glucocorticoids increased side effects increased, therefore, "CYP3A activity levels and associated femoral head necrosis" hypothesis was made, this study aims to initially explore the CYP3A activity in steroid ONFH pathogenesis and clinical for the treatment of the experience of the femoral head necrosis side-bone with effects on CYP3A activity.Observe the influence of PPARr in partial femoral head to investigate the locality fat differentiation. Observe the influence of adiponectin,Osterix and Runx2 to investigate the influence of locality osteoblast, its treatment and prevention of steroid-induced femoral head necrosis provide a theoretical basis.MethodsThirty adult SPF healthy New Zealand rabbits were used, male and female half and half. According to the principle of weight balance, the rabbits were devided into five groups:Control group, Model group, Group inducer, Inhibitor group, JIANGUFANG group. Each group was feed under control and was administrate after one week. The molding method was refer to Yamanoto and the research method and dosage was Toshiaki Masada. In group inducer, the six animals were received daily intramuscular Phenobarbital 25mg/kg, Inhibitor group received oral itraconazole 50 mg/kg every other day to inhibit CYP3A activity; JIANGUFANG group received oral JIANGUFANG Suspension 10ml/kg, Drug content was 0.719g/ml every day and the other six animals were given with saline 10ml/kg as a control.Weight each of the animal for three consecutive weeks, then intramuscular injection of methylprednisolone sodium succinate 40mg/kg for 3 days in the left thight of the rabbits, continue to feed 3 weeks.Before the Experiment or three weeks later, collection 4ml vein blood from the rabbit ear after 8-hour fasting, mixed with Lithium heparin, then put it into the Centrifuge,3000RPM,10 minute. Collect the upper yellow liquid into the Centrifuge Tube,-70℃Storage. Use the GENMED quantitative colorimetric detection kit detect the sample. This method is by erythromycin demethylation enzyme reaction system to methyl erythromycin A product of formaldehyde and Nash after the reagents after the change of absorption peak to determine CYP3A activity in liver samples. Adopt double antibody step clip neo-confucianism, enzyme-linked immunosorbent adsorption test (ELISA) to determinate the glucocorticoid levels in plasma. Air embolism execute the rabbit after six weeks,take the left femoral in liquid nitrogen tank, determinate the glucocorticoid, adiponectin PPARγ, Runx2, Osterix in locality femoral head with ELISA, take the right posterior femoral head, remove soft tissue, marked the specimen bag, fix the sample with 10% Formalin, prosecution pathology testing. Analyzed the data with SPSS 13.0 For Windows Statistical software. Representing measurement data by T test with arithmetic mean(x)and Standard Deviation (S). Analyzed necrosis rate by One-Way ANOVA.Abdopted Spearman rank correlation analysis to progress Analysis of Correlation.ResultsRefer to Matsui, the stage division of histopathology in adult rabbit hormone induced ONFH, this study used the pathologic result to evaluate the success of copying the model. Macroscopic observation showed that visible appearance complete, did not see the femoral subchondral collapse or chip, but the color of femoral articular cartilage was dark; HE dyed slice which was observed by optical microscope showed that in addition to 5 in control group only for 0 level of rabbit pathological changes, there were 9 (47.4%) of grade 2 pathological changes and 10(52.6%) of grade 1 in the rest of the 19 used hormone.Before to use of the drug intervention, hepatic CYP3A activity had no statistically significant difference (P=0.253,0.647,0.242,0.262> 0.05)in compared with the control group, model group, Group inducer, inhibitor group and JIANGUFANG group. After 3 weeks, Compared with the control group, the model group difference was statistically significant (t=1.848, P=> 0.05), the enzymatic enzyme activity of CYP3A was obviously improved after phenobarbital injection and differences are statistically significant (t= 3.678, P=0.004 < 0.05), itraconazole capsule inhibited the CYP3A enzyme activity in the inhibitor group,the difference was statistically significant (t=6.268, P=0.001<0.05), JIANGUFANG can also raise the enzyme's activity, the difference has a statistics meaning; (t=3.84, P=0.004<0.05) And the comparison with model set, inducement set, JIANGUFANGset the enzyme activity increases, and the depressant set enzyme activity lower, and the difference all has a statistics meaning; (t=3.82,3.93,2.57, P=0.013,0.09,0.021<0.05)And comparison with inducement set, the activity obviously lowers of the liver CYP3 A enzyme in depressant set, the difference has a statistics meaning (t=3.714, P=0.013<0.05), JIANGUFANG set the CYP3 A Enzyme the activity has to descend, but the difference has no statistics meaning; (t=0.105, P=0.322>0.05)And with the comparison depressant set, the CYP3 A Enzyme the activity in JIANGUFANG set obviously raises, the difference has a statistics meaning. (t=5.60, P=0.02<0.05)Each comparison used self-comparison of medicine, the blank matched control is used a medicine self-comparison, the liver CYP3 A Enzyme live difference has no statistics meaning; (t=1.859, P=0.122>0.05) The model set is used the medicine the liver CYP3 A Enzyme activity more than before, the difference has no statistics meaning; (t=1.92, P=0.097>0.05)The inducement set is used the medicine the liver CYP3 A Enzyme activity obviously higher than before, the difference has a statistics meaning; (t=5.06, P=0.003<0.05)The depressant set is used the medicine the liver CYP3 A Enzyme activity more lower than before, the difference has a statistics meaning;(t=7.482, P=0.002<0.05)The JIANGUFANG set is used the medicine the liver CYP3 A Enzyme activity more obviously higher than before, the difference has a statistics meaning. (t=5.059, P=0.003<0.05)Just before making a mold with glucocorticoid, Blank control group, model group, induced dose group, inhibitor group, JIANGUFANG plasma glucocorticoid levels not significantly different (the P value distinguishes to 0.952,0.18, 0.099,0.351>0.05), the set adopts a single factor variance to analyze to show F=1.289, P=0.301>0.05 difference have no the statistics;With the medicine interfere after 3 weeks, the set adopts a single factor variance analytical show F=26.72,P=0.001<0.05 shows significant differences between groups, The test of homogeneity of variance, P=0.076>0.05, Show that the homogeneity of variance. Use the using LSD pairwise comparison method, The results showed that compared with the control group, blood plasma sugar skin quality hormone level model set goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics meaning(P=0.527>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also obviously lower, but the difference have no the statistics meaning;(P=0.124>0.05)And model set comparison, the blood plasma level glucocorticoid in inducement set, JIANGUFANG set all lower, the depressant set raise, the difference all has a statistics meaning;(P=0.0001,0.0001,0.003<0.05)And comparison in inducement set, depressant set blood plasma glucocorticoid level goes up obviously, the difference has a statistics meaning(P=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.346>0.05)And depressant set comparison, the JIANGUFANG set obviously lower, the difference has a statistics to learn meaning. (P=0.001<0.05)The data of partial s glucocorticoid content of each bone, the set adopts a single factor variance analytical show F=143.8, P=0.001<0.05 difference shows obvious, through data examination, P=0.494>0.05, express that the data equal. Use the LSD method carries to compare with blank matched control comparison, partial glucocorticoid level model set obviously goes up, the difference has a statistics meaning(P=0.001<0.05) and inducement the set has to lower, difference have no the statistics learn the meaning(P=0.059>0.05), depressant set obviously goes up, the difference has a statistics to learn the meaning(P=0.0001<0.05), JIANGUFANG set also obviously lowers, but difference have no the statistics learn meaning;(P=0.324>0.05)And model set comparison, partial set, JIANGUFANG set of sugar skin quality hormone level inducement all lowers, the depressant set goes up, the difference all has a statistics-to learn meaning; (P=0.0001,0.0001,0.001<0.05) And inducement set comparison, the depressant set the bone partial sugar the skin quality hormone level goes up obviously, the difference has a statistics to learn meaning (p=0.0001<0.05), and the JIANGUFANG set has to descend, but the difference has no statistics meaning;(P=0.331>0. 05)And depressant set comparison, the JIANGUFANG set obviously lowers, the difference has a statistics meaning. (P=0.001<0.05)The liver CYP3 A enzyme activity and the bone partial glucocorticoid level presents to show negative related. (RR value is-0.657, P=0.001)The partial fat allied plain content data of each bone, the set adopts a single factor variance analytical show F=15.78, P=0.001<0.05 difference shows Obvious, through homogeneity of variance test, P=0.102>0.05, express that the variance adopts together the LSD method carries to compare, and blank matched control comparison, the partial fat unites plain level model, the set obviously lowers, the difference has a statistics to learn a meaning(P=0.005<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=0.163>0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also has to lower, but difference have no the statistics learn meaning;(P=0.464>0.05)And model set comparison, partial fat allied plain level inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.0001,0.013, 0.023<0.05) And inducement set comparison, the depressant set the bone partial fat allied plain level lowers obviously, the difference has a statistics meaning(P=0.0001<0.05), the JIANGUFANG set has to lower, and the difference includes a statistics meaning; (P=0.041m<0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)The data of partial content of PPARγof each bone, the set adopts a single factor variance analytical show F=91.79, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.001<0.05, express that the variance adopts not and together the Tamhane method carries on compare, and blank matched control comparison, the partial PPAR theγlevel model set obviously goes up, the difference has a statistics to learn a meaning(P=0.018<0.05) and inducement the set has to go up, difference have no the statistics learn the meaning(P=1.00>0.05), depressant set obviously goes up, the difference has a statistics meaning(P=0.0001<0.05), JIANGUFANG set also slightly has to go up, but difference have no the statistics learn meaning; (P=1.00>0.05)And model set comparison, partial PPAR the set, JIANGUFANG set of theγlevel inducement all lowers, the depressant set obviously goes up, the difference all has a statistics meaning; (P=0.045,0.006, 0.033<0.05)And inducement set comparison, depressant set the bone partial level of PPARγobviously goes up, the difference has a statistics meaning (P=0.002<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.891>0.05)And depressant set comparison, the JIANGUFANG set obviously lowers Gao, the difference has a statistics meaning. (P=0.001<0.05)The partial Runx2 content data of each bone, the set adopts a single factor variance analytical show F=88.4, P=0.001<0.05 difference shows obvious, the test of homogeneity of variance, P=0.003<0.05, express that the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Runx2 content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.0031<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.013<0.05), depressant set obviously lowers, the difference has a statistics meaning (P=0.0001<0.05), JIANGUFANG set also obviously goes up, the difference has a statistics meaning;(P=0.002<0.05)And model set comparison, bone partial Runx2 content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning; (P=0.003,0.001,0.001<0.05) And inducement set comparison, depressant set the bone partial Runx2 contents lower obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to lower, and the difference has no statistics meaning;(P=0.288>0.05)And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.001<0.05)Bone partial Osterix content set adopts a single factor variance analytical show F=38.07, P=0.001<0.05 difference shows Obvious, the test of homogeneity of variance, P=0.002<0.05, the variance adopts not and together the Tamhane method carries to compare, and blank matched control comparison, bone partial Osterix content model the set obviously lowers, the difference has a statistics to learn a meaning(P=0.037<0.05) and inducement the set obviously goes up, the difference includes a statistics meaning(P=0.006<0.05), depressant set obviously lowers, the difference has a statistics meaning(P=0.004<0.05), JIANGUFANG set also obviously goes up, but difference have no the statistics learn meaning; (P=0.066>0.05) And model set comparison, bone partial Osterix content inducement the set, JIANGUFANG set all goes up, the depressant set obviously lowers, the difference all has a statistics meaning;(P=0.001,0.025,0.022<0.05)And inducement set comparison, the depressant set the bone partial Osterix content lowers obviously, the difference has a statistics meaning(P=0.001<0.05), the JIANGUFANG set has to go up, and the difference has no statistics meaning; (P=0.76>0.05) And depressant set comparison, the JIANGUFANG set obviously goes up, the difference has a statistics meaning. (P=0.012<0.05)Each bad dead area with bad dead rate data, use a single factor variance analysis(One-Way ANOVA), the statistics shows:Through Levene variance examines, the variance is together;(P=0.091 and P=0.151)Together adopt a LSD examination method under the variance, the bad dead area result shows:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.037)Model set and depressant set comparison, both the bad dead area shows the Obvious difference P<0.05;(P=0.023)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set set bad dead area lowers, both have already shown the Obvious difference P<0.05;(P=0.015)And inducement set comparison, the depressant set bad dead area obviously goes up, the difference has a statistics meaning P<0.05(P=0.012), the JIANGUFANG set bad dead area has to go up, but the difference don't show Obvious P>0.05;(P=0.423)And depressant set, the JIANGUFANG set bad dead area obviously lowers, P<0.05; (P=0.034) The bad dead rate shows as a result:Model set and inducement set comparison, to compare with model set, the inducement the set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.023)Model set and depressant set comparison, both the bad dead rate shows the Obvious difference P<0.05; (P=0.044)Model set and JIANGUFANG set comparison, to compare with model set, the JIANGUFANG set bad dead rate lowers, both have already shown the Obvious difference P<0.05;(P=0.024)And inducement set comparison, the depressant set bad dead rate obviously goes up, the difference has a statistics meaning P<0.05(P=0.016), the JIANGUFANG set bad dead rate has to go up, but the difference don't show Obvious P>0.05;(P=0.623)And depressant set, the JIANGUFANG set bad dead rate obviously lowers, P<0.05. (P=0.025)The liver CYP3 enzyme activity and the bone bad dead area, bad dead rate all become to show Obvious of negative related.ConclusionsThis research result expresses the activity of The liver CYP3A enzyme presents with the occurrence of the hormone femoral head necrosis to show Obvious negative the related①liver CYP3 A Mao activity increase Gao to make a bone partial glucocorticoid content reduced.②The liver CYP3 enzyme activity increases Gao to lower hormone the bone bad dead lead, reduce bad dead area and ease pathologic ratings. This may be The liver CYP3 enzyme can outward the source glucocorticoid contain clearance function and make glucocorticoid shortenned in time of storing of bone part, the density lower and lowered the incidence rate of the bone bad dead thus. The experiment research of the influence of Chinese herbal medicine JIANGUFANG upon The liver CYP3 enzyme and hormone ONFH expresses that①JIANGUFANG can strengthen the activity of The liver CYP3 enzyme as a result.②JIANGUFANG can prevent°fro the occurrence of the hormone femoral head necrosis, ease bad dead pathologic degree.③JIANGUFANG prevents and cures the occurrence of the hormone femoral head necrosis to be the level that passes to lower a bone partial PPARγand exaltation Runx2, Osterix and the fat allied vegetable and attains to repress into a fat and promotes the function of ossification.④Provide an experiment for the Chinese medicine medicine"cure don't disease" theories prevention and cure hormone femoral head necrosis basis.