Effect Of The Curcumae Rhizoma And Sparganii Rhizoma Component Compatibility On Bone Metabolism In Rats With Uterine Leiomyoma

Objective:This experiment uses a combination of two modes（intramuscular injection of estrogen and progesterone,stimulation of external factors）,to established the model.Intervened with Curcumae Rhizoma and Sparganii Rhizoma component compatibility,then observed the progesterone（P）and estradiol（E₂）in serum,transforming growth factor-β1（TGF-β1）,estrogen receptor（ER）,progesterone receptor（PR）in uterine tissue,the expression changes of TGF-β1,estrogen receptorα（ERα）,bone morphogenetic protein（BMP-2）gene and protein in the femur,and bone microstructure.are preliminarily explored the effect of Curcumae Rhizoma and Sparganii Rhizoma component compatibility on the bone metabolism of uterine leiomyoma（UL）and its possible mechanism while effectively treating UL.Method:1.Modeling:48 rats were randomly divided into 6 groups:normal control group,model control group,Yang Yao group（Gongliuxiao capsule group）,S-E group（Sparganii Rhizoma total flavonoids-Curcumae Rhizoma volatile oil group 1:1compatibility group）,S group（Sparganii Rhizoma total flavonoids group）,E group（Curcumae Rhizoma volatile oil group）,two cages per group,4 per cage.They werenumbered and marked with yellow（picric acid）and reared adaptively for 2 weeks.All rats are sensitive,behave actively,have shiny hair,and are in good condition.Except for the normal control group,rats in the other groups were injectedintramuscularly with estradiol injection（0.5mg/kg）into the inner thigh everymorning,and progesterone injection（1mg/mouse）was injected intramuscularlyevery other day every week,three times a week.At the same time,each mouse isinjected with epinephrine injection（0.5mg/kg）subcutaneously once a week.After4 hours of injection every day,one of the following stimuli was randomly given（（1）Day and night reverse,（2）60db noise stimulation for 3h,（3）tail hanging upsidedown for 10min,（4）5℃-10℃swimming in ice water for 4min）for a total of nineweeks.Starting from the sixth week,rats in the Yang Yao group,S-E group,S group,and E group were intragastrically administered with 10ml/kg of interventionmedicine in each group every afternoon after modeling.Rats in the normal controlgroup were intragastrically administered with ultrapure water（2ml/d）.2.Indicator detection:（1）After the experiment,blood was collected from aorta abdominal of rats,and theserum was separated,and the concentration of P and E₂ in the serum was detectedby ELISA.（2）The rat uterus was separated,photographed,weighed and measured.In order todetect the expression level of TGF-β1,ER,PR in uterine tissue,collect appropriateamount of uterine tissue for pathological HE staining and immunohistochemistry.（3）The left femur of the rat was divided into two segments,and a part of the total RNAwas extracted to detect the expression of TGF-β1,ERα,and BMP-2 m RNA in thefemur by fluorescence quantitative PCR.The other part uses Western blot to detectthe protein expression of the above indicators in bone tissue.（4）The right femur of the rat was used for Micro-CT scan to calculate bonemicrostructure parameters:bone mineral density（BMD）,bone volume（BV）,bonesurface area（BS）,Bone surface area over bone volume（BS/BV）,bone volumefraction（BV/TV）,trabecular number（Tb.N）,trabecular thickness（Tb.Th）,Trabecular separation（Tb.Sp）.（5）The bone tissue was decalcified to prepare slices for HE pathological staining toobserve the pathological changes of the distal femur tissue.Result:1.The rats in the normal group are still active and their weight gains fast.The modelrats had irregular fur loss,beard loss,weight loss,and weight gain decreasedsignificantly.The bilateral uterus of the rats in the normal control group weresymmetrical,with bright colors,uniform thickness,and no swelling or nodules.Thepathological results showed that the thickness of the muscle layer was uniform,andthe cells in the propria under the mucosal layer were tightly arranged and the outlinewas clear.Obvious distortions,swelling,and nodules can be seen in the appearanceof the uterus of the rats in the model group,and the transverse and vertical diametersof the uterus are significantly increased.The pathological results showed that a largenumber of smooth muscle cells proliferated in the propria,a small number ofeosinophils and mucosal epithelial cells proliferated,arranged in multiple layers.2.In the serum,the concentration of P and E₂ in the model group（p<0.05）,and thedifference was statistically significant.Compared with the model group,there wasa significant difference in the reduction of P concentration in the S-E group（p<0.05）,and the concentration of E₂ in the Yang Yao group,S-E group,and E group wassignificantly reduced（p<0.05）.In the results of immunohistochemistry,theexpression levels of TGF-β1（p<0.001）,ER（p<0.01）,and PR（p<0.05）in the uterinetissue of the model group were significantly higher than those of the normal controlgroup.The concentrations of TGF-β1,ER,and PR in the positive medicine group（p<0.01）,S-E group（p<0.01）,and E group（p<0.05）were significantly reduced tocompared with the model group,with statistical differences.These data all showthat the S-E group has a good therapeutic effect on UL.3.In the Western Blot test results,compared with the normal control group,the proteinexpression levels of TGF-β1,ERα,and BMP-2 in the model group were notstatistically different.There was a significant increase in TGF-β1 in the positivemedicine group and the S-E group（p<0.05）.The expression level of ERαproteinshowed a downward trend in the model group and an upward trend in theadministration group,but there was no statistical difference.The protein expressionlevel of BMP-2 in the femur of rats in all experimental groups did not change much.4.In the PCR detection and analysis,the expression levels of TGF-β1,ERα,and BMP-2 m RNA in the femur were only numerically changed among all the experimentalgroups,and there was no statistical difference（p>0.05）,but the S-E group hadnumerical results suggest that the Curcumae Rhizoma and Sparganii Rhizomacomponent compatibility can increase the levels of TGF-β1,ERα,and BMP-2m RNA in rat femurs.5.Micro-CT results showed that BMD values of femur in model group（P<0.001）,Yang Yao group（P<0.01）,S-E group（P<0.01）,s group（P<0.001）and E group（P<0.001）were significantly decreased.The effect of increasing the BMD valueof the Yang Yao group and the S-E group was better than that of the S group and theE group（p>0.05）.There was no difference in BV in the model group（p>0.05）.TheBV value of the positive medicine group（p<0.001）,the S-E group（p<0.001）,the Sgroup（p<0.01）,and the E group（p<0.01）decreased.There was no significantchange among BS,BS/BV,and BV/TV groups.In the S-E group,the value of Tb.Nwas higher than that of the normal control group（p<0.05）,and the value of Tb.Thwas significantly reduced（p<0.01）.There was no significant difference in the valueof Tb.Sp,but there was a decreasing trend.6.The results of HE staining of the distal femur bone tissue showed that the bonetissues of all experimental groups.The cancellous bone is composed of a largenumber of needle-shaped bone trabeculae.The trabeculae were connected with eachother to form a porous network structure.The bone marrow filled the cavity,thecells were dense,the morphology and structure were complete,and no obviousabnormality was found.Conclusion:1.Curcumae Rhizoma and Sparganii Rhizoma component compatibility can reducethe serum concentration of P and E₂ with UL and the high expression of TGF-β1,ER,PR in uterine tissues,and effectively treat UL.2.Curcumae Rhizoma and Sparganii Rhizoma component compatibility can reducethe expression level of TGF-β1 and ER in the uterus of rats with UL,and has noadverse effect on the expression of TGF-β1,ERα,BMP-2 m RNA and protein in thefemur.3.UL can affect the bone density and bone volume of rats,which may be related tothe influence on the weight gain of rats.The Curcumae Rhizoma and SparganiiRhizoma component compatibility can increase the decreased bone density,increasethe number of trabecular bones,and increase bone Trabecular thickness reduces theseparation of trabecular bone.4.Curcumae Rhizoma and Sparganii Rhizoma component compatibility caneffectively treat rats with UL and has no adverse effect on bone metabolism.