基于Runx2相关基因表达级联调控对糖皮质激素性骨质疏松症大鼠肾虚证候病机的研究
摘要
目的:
通过体内实验,基于成骨分化特异调控基因Runx2相关基因表达级联调控,探讨糖皮质激素性骨质疏松症(glucocorticoid-induced osteoporosis,GIOP)肾虚病机的微观发病机制,以及补肾益髓中药复方的疗效机理,并为进一步阐明中医“肾藏精生髓主骨”理论的科学内涵提供实验依据。
材料与方法:
本研究采用后肢肌注地塞米松(2.5mg/kg,每周2次,连续9周)的方法复制GIOP大鼠模型,选用笔者导师郑洪新教授在中医“肾藏精生髓主骨”理论指导下的多年科研、临床实践之补肾益髓中药复方(主要由鹿茸、淫羊藿、牡蛎组成,功效:补肾填精、益髓壮骨)作为实验因素,选用补中益气颗粒(功效:补中益气)和血府逐瘀胶囊(功效:活血祛瘀)作为对照药,选用骨疏康颗粒(功效:补肾益气、活血壮骨)作为阳性对照药,以骨、肾组织Runx2、Runx2的上游调控基因Msx2、Dlx5、Runx2的下游靶基因Osterix mRNA及蛋白表达为效应指标,进行体内实验。实验大鼠按体重分层随机分为正常组、模型空白组、补肾益髓中药复方组、补中益气颗粒组、血府逐瘀胶囊组、骨疏康颗粒阳性对照组。灌胃给药9周。实验期间,每天观察大鼠皮毛的状态、活动状态、反应状态、是否有聚堆、是否有弓腰塌背的体态改变以及食量、大便状态等,并做好记录。9周后处死动物,取股骨、肾组织检测以下指标:应用XR-26型双能X线骨密度仪测定股骨骨密度,应用IX71FL型倒置荧光显微镜观察股骨头石蜡切片HE染色显微形态结构,以评价动物模型的成立及药物疗效;实时定量RT-PCR(Real-Time Quantitative RT-PCR)检测骨、肾组织Msx2、Dlx5、Runx2、Osterix mRNA表达,Western Blot检测骨、肾组织Msx2、Dlx5、Runx2、Osterix蛋白表达。
结果:
1.大鼠生物学表征信息:正常组大鼠实验全程皮毛有光泽,活泼喜动,反应灵敏,无竖毛、聚堆、弓腰塌背等表现,食欲佳,食量、大便正常;造模9周结束时,模型空白组大鼠均呈现明显的弓腰塌背体态,皮毛无光泽,竖毛,喜静懒动,萎靡不振,反应迟钝,喜蜷缩聚堆,食欲下降,便软等肾虚表现;各给药组大鼠的弓腰塌背等肾虚表现均有不同程度的改善,其中补肾益髓中药复方组和骨疏康颗粒阳性对照组改善较明显,以补肾益髓中药复方组改善最明显。
2.大鼠离体股骨骨密度(BMD):与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显升高(P<0.01),骨疏康颗粒阳性对照组明显升高(P<0.05),补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
3.大鼠股骨头石蜡切片HE染色显微形态结构:光镜下可见,正常组骨小梁粗壮、饱满,形态结构完整,余留骨重建空间较少,骨髓腔相对较小;与正常组比较,模型空白组骨小梁明显变细,形态结构完整性差,有些部位破坏、断裂,余留骨重建空间较多,骨髓腔明显扩大;与模型空白组比较,补肾益髓中药复方组和骨疏康颗粒阳性对照组骨小梁形态结构明显改善,骨小梁增粗,结构较紧密,余留骨重建空间减少,骨髓腔减小,尤以补肾益髓中药复方组改善更为明显,而补中益气颗粒组和血府逐瘀胶囊组骨小梁形态结构虽有所改善,但不明显。
4. Real-Time Quantitative RT-PCR结果:
(1)骨组织:正常大鼠骨组织可检测到Msx2、Dlx5、Runx2、Osterix mRNA表达。
①Msx2 mRNA表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix mRNA表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
(2)肾组织:正常大鼠肾组织可检测到Msx2、Dlx5、Runx2、Osterix mRNA表达。
①Msx2 mRNA表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix mRNA表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
5. Western Blot结果:
(1)骨组织:正常大鼠骨组织可检测到Msx2、Dlx5、Runx2、Osterix蛋白表达。
①Msx2蛋白表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix蛋白表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
(2)肾组织:正常大鼠肾组织可检测到Msx2、Dlx5、Runx2、Osterix蛋白表达。
①Msx2蛋白表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix蛋白表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
结论:
1.肌肉注射地塞米松(2.5mg/kg,每周2次,连续9周)的方法可以成功复制GIOP大鼠模型,GIOP的证候病机属肾虚,补肾益髓中药复方可有效防治该病,疗效明显优于健脾中药和活血中药。
2.正常大鼠骨、肾组织存在Runx2、Osterix、Msx2、Dlx5 mRNA和蛋白表达;肾藏精生髓可能通过级联调控肾系统之骨与肾Runx2相关基因表达而实现主骨。
3.长期、超生理剂量应用GC耗损肾精,精亏髓少,骨、肾失养,功能失常,Runx2相关基因表达级联失调:骨组织Runx2、Osterix、Dlx5 mRNA及蛋白表达降低,Msx2 mRNA及蛋白表达升高;肾组织Runx2、Osterix、Dlx5 mRNA及蛋白表达升高,Msx2 mRNA及蛋白表达降低,可能是GIOP肾虚病机的微观发病机制之一。
4.补肾益髓中药复方功能补肾填精、益髓壮骨,可恢复肾“藏精生髓主骨”的功能,进而级联调节肾系统之骨与肾Runx2相关基因表达:上调骨组织Runx2、Osterix、Dlx5mRNA及蛋白表达,下调骨组织Msx2 mRNA及蛋白表达;下调肾组织Runx2、Osterix、Dlx5 mRNA及蛋白表达,上调肾组织Msx2 mRNA及蛋白表达,可能是补肾益髓中药复方防治GIOP的疗效机理之一。
Purpose:
To discuss the microcosmic pathogenesis of glucocorticoid-induced osteoporosis (GIOP) of kidney deficiency and the therapeutic effect and mechanism of reinforcing the kidney to replenish marrow traditional Chinese herbs compound based on cascading regulation of Runx2 interrelated genes expression through in vivo experiment,so as to provide experimental evidence for exploring the further scientific connotation of the traditional Chinese medicine (TCM) theory“the kidney storing essence to produce marrow to dominate bones”.
Materials and methods:
In the vivo experiment,we established the GIOP rat models by hind limb intramuscular injection of dexamethasone(2.5mg/kg,twice a week,9 weeks continuously),chose the writer’s tutor - Prof.ZHENG Hong-xin’s reinforcing the kidney to replenish marrow traditional Chinese herbs compound (mainly consisting of pilose antler, epimedium and oyster, function: reinforcing the kidney to replenish essence, tonifying marrow to strengthen the bone) of years of scientific research and clinical practice under the guidance of the TCM theory”the kidney storing essence to produce marrow to dominate bones”as experimental factors,chose reinforcing the middle to benefit qi granula with the function of reinforcing the middle to benefit qi and removing blood-house stasis capsule with the function of removing stasis by promoting blood circulation as control drug,chose GUSHUKANG granula with the function of reinforcing the kidney to benefit qi, promoting blood circulation to strengthen the bone as positive control drug and took mRNA and protein expression of Msx2,Dlx5,Runx2 and Osterix in bone and kidney tissues as effective indicators.We stratified all the experimental rats by different body weight and then divided them randomly into normal group,blank model group,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group,reinforcing the middle to benefit qi granula group,removing blood-house stasis capsule group and GUSHUKANG granula positive control group.We administrated them by gavage for 9 weeks. During the experiment,we observed the rats’state of fur, activity, reaction,stool,food intake and whether they gathered together and had stoop and fallen back posture and made a record everyday.After 9 weeks,we put all the rats to death and get their femur and kidney to detect the following indicators:We detected the femur bone mineral density with the XR-26 dual energy X-ray absorptiometry and observed the micro-morphology structure of HE stained paraffin sections of femoral heads with the IX71FL inverted fluorescence microscope to evaluate the establishment of the animal model and the therapeutic effect of the drugs.We detected mRNA expression of Msx2,Dlx5,Runx2 and Osterix in bone and kidney tissues by Real-Time Quantitative RT-PCR and detected their protein expression by Western Blot.
Results:
1. Rats’biological manefestation information:During the whole experiment period, normal group rats with shiny fur, good appetite and normal food intake and stool were lively,active and responsive,without such manifestations as pilo-erection, huddling and gathering together and posture of stoop and fallen back etc.At the end of 9 weeks of modeling, blank model group rats showed significant manifestations of kidney deficiency such as posture of stoop and fallen back, dull fur, pilo-erection, quiet, cachexia, unresponsive, huddling and gathering together, loss of appetite and soft feces etc.The manifestation of kidney deficiency such as posture of stoop and fallen back etc of every administrated group rats had different degrees of improvement, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group and GUSHUKANG granula positive control group had more significant improvement and reinforcing the kidney to replenish marrow traditional Chinese herbs compound group had the most significant improvement.
2. Rats’vitro femur bone mineral density (BMD):Compared with normal group,blank model group decreased significantly(P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group increased significantly(P<0.01),GUSHUKANG granula positive control group increased significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically (P>0.05).
3. Micro-morphology structure of HE stained paraffin sections of rats’femoral heads:We observed that normal group rats’bone trabeculas were thick and full, with morphological integrity and less remaining bone remodeling space,and the bone marrow cavities were relatively small with the light microscope.Compared with normal group,blank model group rats’bone trabeculas were thinner obviously,with bad morphological integrity of some damaged and fractured parts and more remaining bone remodeling space,and the bone marrow cavities enlarged obviously.Compared with blank model group,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group and GUSHUKANG granula positive control group rats’bone trabeculas morphology structure improved obviously,with bone trabeculas thickening, structure more compact,remaining bone remodeling space lessening and the bone marrow cavities reducing,in particular,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group improved more obviously,while reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group rats’bone trabeculas morphology structure improved at a certain degree,not obviously.
4. Results of Real-Time Quantitative RT-PCR
(1) Bone tissues: Msx2, Dlx5, Runx2, Osterix mRNA expression can be detected in normal rats’bone tissues.
①Msx2 mRNA expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P < 0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix mRNA expression:Compared with normal group,blank model group decreased significantly (P< 0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
(2) Kidney tissues: Msx2, Dlx5, Runx2, Osterix mRNA expression can be detected in normal rats’kidney tissues.
①Msx2 mRNA expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P < 0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix mRNA expression:Compared with normal group,blank model group increased significantly (P< 0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
5. Results of Western Blot
(1) Bone tissues: Msx2, Dlx5, Runx2, Osterix protein expression can be detected in normal rats’bone tissues.
①Msx2 protein expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix protein expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
(2) Kidney tissues: Msx2, Dlx5, Runx2, Osterix protein expression can be detected in normal rats’kidney tissues.
①Msx2 protein expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix protein expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
Conclusions:
1. The method of intramuscular injection of dexamethasone(2.5mg/kg,twice a week,9 weeks continuously) can establish GIOP rat models successfully.The syndrome pathogenesis of GIOP belongs to kidney deficiency and reinforcing the kidney to replenish marrow traditional Chinese herbs compound can prevent and treat GIOP effectively,which has more evident preventative and therapeutic effect on GIOP than the strengthening the spleen herbs and the promoting blood circulation herbs.
2. Normal rats express Runx2,Osterix,Msx2 and Dlx5 at mRNA and protein level in bone tissues and kidney tissues.The kidney storing essence to produce marrow to dominate bones by cascading regulation of Runx2 interrelated genes expression in bone and kidney of kidney system.
3. Long-term and super-physiological dose usage of GC consumes kidney essence, leading to deficiency of kidney essence and bone marrow unable to moisten and nourish bones and kidney,resulting in dysfunction of bones and kidney incapable of regulating Runx2 interrelated genes expression cascadingly.Decrease of Runx2,Osterix,and Dlx5 mRNA and protein expression and increase of Msx2 mRNA and protein expression in bone tissues.Increase of Runx2,Osterix,and Dlx5 mRNA and protein expression and decrease of Msx2 mRNA and protein expression in kidney tissues.Maybe the above-mentioned is one aspect of the microcosmic pathogenesis of GIOP of kidney deficiency.
4. The reinforcing the kidney to replenish marrow traditional Chinese herbs compound with the function of reinforcing the kidney to replenish essence, tonifying marrow to strengthen the bone can recover the kidney’s function of storing essence to produce marrow to dominate bones to regulate Runx2 interrelated genes expression in bone and kidney of kidney system cascadingly.Up-regulating Runx2,Osterix,and Dlx5 mRNA and protein expression and down-regulating Msx2 mRNA and protein expression in bone tissues.Down-regulating Runx2,Osterix,and Dlx5 mRNA and protein expression and up-regulating Msx2 mRNA and protein expression in kidney tissues.Maybe the above-mentioned is one of the mechanisms of reinforcing the kidney to replenish marrow traditional Chinese herbs compound to prevent and treat GIOP.
通过体内实验,基于成骨分化特异调控基因Runx2相关基因表达级联调控,探讨糖皮质激素性骨质疏松症(glucocorticoid-induced osteoporosis,GIOP)肾虚病机的微观发病机制,以及补肾益髓中药复方的疗效机理,并为进一步阐明中医“肾藏精生髓主骨”理论的科学内涵提供实验依据。
材料与方法:
本研究采用后肢肌注地塞米松(2.5mg/kg,每周2次,连续9周)的方法复制GIOP大鼠模型,选用笔者导师郑洪新教授在中医“肾藏精生髓主骨”理论指导下的多年科研、临床实践之补肾益髓中药复方(主要由鹿茸、淫羊藿、牡蛎组成,功效:补肾填精、益髓壮骨)作为实验因素,选用补中益气颗粒(功效:补中益气)和血府逐瘀胶囊(功效:活血祛瘀)作为对照药,选用骨疏康颗粒(功效:补肾益气、活血壮骨)作为阳性对照药,以骨、肾组织Runx2、Runx2的上游调控基因Msx2、Dlx5、Runx2的下游靶基因Osterix mRNA及蛋白表达为效应指标,进行体内实验。实验大鼠按体重分层随机分为正常组、模型空白组、补肾益髓中药复方组、补中益气颗粒组、血府逐瘀胶囊组、骨疏康颗粒阳性对照组。灌胃给药9周。实验期间,每天观察大鼠皮毛的状态、活动状态、反应状态、是否有聚堆、是否有弓腰塌背的体态改变以及食量、大便状态等,并做好记录。9周后处死动物,取股骨、肾组织检测以下指标:应用XR-26型双能X线骨密度仪测定股骨骨密度,应用IX71FL型倒置荧光显微镜观察股骨头石蜡切片HE染色显微形态结构,以评价动物模型的成立及药物疗效;实时定量RT-PCR(Real-Time Quantitative RT-PCR)检测骨、肾组织Msx2、Dlx5、Runx2、Osterix mRNA表达,Western Blot检测骨、肾组织Msx2、Dlx5、Runx2、Osterix蛋白表达。
结果:
1.大鼠生物学表征信息:正常组大鼠实验全程皮毛有光泽,活泼喜动,反应灵敏,无竖毛、聚堆、弓腰塌背等表现,食欲佳,食量、大便正常;造模9周结束时,模型空白组大鼠均呈现明显的弓腰塌背体态,皮毛无光泽,竖毛,喜静懒动,萎靡不振,反应迟钝,喜蜷缩聚堆,食欲下降,便软等肾虚表现;各给药组大鼠的弓腰塌背等肾虚表现均有不同程度的改善,其中补肾益髓中药复方组和骨疏康颗粒阳性对照组改善较明显,以补肾益髓中药复方组改善最明显。
2.大鼠离体股骨骨密度(BMD):与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显升高(P<0.01),骨疏康颗粒阳性对照组明显升高(P<0.05),补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
3.大鼠股骨头石蜡切片HE染色显微形态结构:光镜下可见,正常组骨小梁粗壮、饱满,形态结构完整,余留骨重建空间较少,骨髓腔相对较小;与正常组比较,模型空白组骨小梁明显变细,形态结构完整性差,有些部位破坏、断裂,余留骨重建空间较多,骨髓腔明显扩大;与模型空白组比较,补肾益髓中药复方组和骨疏康颗粒阳性对照组骨小梁形态结构明显改善,骨小梁增粗,结构较紧密,余留骨重建空间减少,骨髓腔减小,尤以补肾益髓中药复方组改善更为明显,而补中益气颗粒组和血府逐瘀胶囊组骨小梁形态结构虽有所改善,但不明显。
4. Real-Time Quantitative RT-PCR结果:
(1)骨组织:正常大鼠骨组织可检测到Msx2、Dlx5、Runx2、Osterix mRNA表达。
①Msx2 mRNA表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix mRNA表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
(2)肾组织:正常大鼠肾组织可检测到Msx2、Dlx5、Runx2、Osterix mRNA表达。
①Msx2 mRNA表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix mRNA表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
5. Western Blot结果:
(1)骨组织:正常大鼠骨组织可检测到Msx2、Dlx5、Runx2、Osterix蛋白表达。
①Msx2蛋白表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix蛋白表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
(2)肾组织:正常大鼠肾组织可检测到Msx2、Dlx5、Runx2、Osterix蛋白表达。
①Msx2蛋白表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药复方组明显上调(P<0.01),骨疏康颗粒阳性对照组明显上调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05)。
②Dlx5、Runx2、Osterix蛋白表达:与正常组比较,模型空白组明显升高(P<0.01);与模型空白组比较,补肾益髓中药复方组明显下调(P<0.01),骨疏康颗粒阳性对照组明显下调(P<0.05);补中益气颗粒组和血府逐瘀胶囊组虽有降低趋势,但无统计学意义(P>0.05)。
结论:
1.肌肉注射地塞米松(2.5mg/kg,每周2次,连续9周)的方法可以成功复制GIOP大鼠模型,GIOP的证候病机属肾虚,补肾益髓中药复方可有效防治该病,疗效明显优于健脾中药和活血中药。
2.正常大鼠骨、肾组织存在Runx2、Osterix、Msx2、Dlx5 mRNA和蛋白表达;肾藏精生髓可能通过级联调控肾系统之骨与肾Runx2相关基因表达而实现主骨。
3.长期、超生理剂量应用GC耗损肾精,精亏髓少,骨、肾失养,功能失常,Runx2相关基因表达级联失调:骨组织Runx2、Osterix、Dlx5 mRNA及蛋白表达降低,Msx2 mRNA及蛋白表达升高;肾组织Runx2、Osterix、Dlx5 mRNA及蛋白表达升高,Msx2 mRNA及蛋白表达降低,可能是GIOP肾虚病机的微观发病机制之一。
4.补肾益髓中药复方功能补肾填精、益髓壮骨,可恢复肾“藏精生髓主骨”的功能,进而级联调节肾系统之骨与肾Runx2相关基因表达:上调骨组织Runx2、Osterix、Dlx5mRNA及蛋白表达,下调骨组织Msx2 mRNA及蛋白表达;下调肾组织Runx2、Osterix、Dlx5 mRNA及蛋白表达,上调肾组织Msx2 mRNA及蛋白表达,可能是补肾益髓中药复方防治GIOP的疗效机理之一。
Purpose:
To discuss the microcosmic pathogenesis of glucocorticoid-induced osteoporosis (GIOP) of kidney deficiency and the therapeutic effect and mechanism of reinforcing the kidney to replenish marrow traditional Chinese herbs compound based on cascading regulation of Runx2 interrelated genes expression through in vivo experiment,so as to provide experimental evidence for exploring the further scientific connotation of the traditional Chinese medicine (TCM) theory“the kidney storing essence to produce marrow to dominate bones”.
Materials and methods:
In the vivo experiment,we established the GIOP rat models by hind limb intramuscular injection of dexamethasone(2.5mg/kg,twice a week,9 weeks continuously),chose the writer’s tutor - Prof.ZHENG Hong-xin’s reinforcing the kidney to replenish marrow traditional Chinese herbs compound (mainly consisting of pilose antler, epimedium and oyster, function: reinforcing the kidney to replenish essence, tonifying marrow to strengthen the bone) of years of scientific research and clinical practice under the guidance of the TCM theory”the kidney storing essence to produce marrow to dominate bones”as experimental factors,chose reinforcing the middle to benefit qi granula with the function of reinforcing the middle to benefit qi and removing blood-house stasis capsule with the function of removing stasis by promoting blood circulation as control drug,chose GUSHUKANG granula with the function of reinforcing the kidney to benefit qi, promoting blood circulation to strengthen the bone as positive control drug and took mRNA and protein expression of Msx2,Dlx5,Runx2 and Osterix in bone and kidney tissues as effective indicators.We stratified all the experimental rats by different body weight and then divided them randomly into normal group,blank model group,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group,reinforcing the middle to benefit qi granula group,removing blood-house stasis capsule group and GUSHUKANG granula positive control group.We administrated them by gavage for 9 weeks. During the experiment,we observed the rats’state of fur, activity, reaction,stool,food intake and whether they gathered together and had stoop and fallen back posture and made a record everyday.After 9 weeks,we put all the rats to death and get their femur and kidney to detect the following indicators:We detected the femur bone mineral density with the XR-26 dual energy X-ray absorptiometry and observed the micro-morphology structure of HE stained paraffin sections of femoral heads with the IX71FL inverted fluorescence microscope to evaluate the establishment of the animal model and the therapeutic effect of the drugs.We detected mRNA expression of Msx2,Dlx5,Runx2 and Osterix in bone and kidney tissues by Real-Time Quantitative RT-PCR and detected their protein expression by Western Blot.
Results:
1. Rats’biological manefestation information:During the whole experiment period, normal group rats with shiny fur, good appetite and normal food intake and stool were lively,active and responsive,without such manifestations as pilo-erection, huddling and gathering together and posture of stoop and fallen back etc.At the end of 9 weeks of modeling, blank model group rats showed significant manifestations of kidney deficiency such as posture of stoop and fallen back, dull fur, pilo-erection, quiet, cachexia, unresponsive, huddling and gathering together, loss of appetite and soft feces etc.The manifestation of kidney deficiency such as posture of stoop and fallen back etc of every administrated group rats had different degrees of improvement, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group and GUSHUKANG granula positive control group had more significant improvement and reinforcing the kidney to replenish marrow traditional Chinese herbs compound group had the most significant improvement.
2. Rats’vitro femur bone mineral density (BMD):Compared with normal group,blank model group decreased significantly(P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group increased significantly(P<0.01),GUSHUKANG granula positive control group increased significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically (P>0.05).
3. Micro-morphology structure of HE stained paraffin sections of rats’femoral heads:We observed that normal group rats’bone trabeculas were thick and full, with morphological integrity and less remaining bone remodeling space,and the bone marrow cavities were relatively small with the light microscope.Compared with normal group,blank model group rats’bone trabeculas were thinner obviously,with bad morphological integrity of some damaged and fractured parts and more remaining bone remodeling space,and the bone marrow cavities enlarged obviously.Compared with blank model group,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group and GUSHUKANG granula positive control group rats’bone trabeculas morphology structure improved obviously,with bone trabeculas thickening, structure more compact,remaining bone remodeling space lessening and the bone marrow cavities reducing,in particular,reinforcing the kidney to replenish marrow traditional Chinese herbs compound group improved more obviously,while reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group rats’bone trabeculas morphology structure improved at a certain degree,not obviously.
4. Results of Real-Time Quantitative RT-PCR
(1) Bone tissues: Msx2, Dlx5, Runx2, Osterix mRNA expression can be detected in normal rats’bone tissues.
①Msx2 mRNA expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P < 0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix mRNA expression:Compared with normal group,blank model group decreased significantly (P< 0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
(2) Kidney tissues: Msx2, Dlx5, Runx2, Osterix mRNA expression can be detected in normal rats’kidney tissues.
①Msx2 mRNA expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P < 0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix mRNA expression:Compared with normal group,blank model group increased significantly (P< 0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
5. Results of Western Blot
(1) Bone tissues: Msx2, Dlx5, Runx2, Osterix protein expression can be detected in normal rats’bone tissues.
①Msx2 protein expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix protein expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P < 0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
(2) Kidney tissues: Msx2, Dlx5, Runx2, Osterix protein expression can be detected in normal rats’kidney tissues.
①Msx2 protein expression:Compared with normal group,blank model group decreased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group up-regulated significantly(P<0.01),GUSHUKANG granula positive control group also up-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to increase,but neither had significant difference statistically(P>0.05).
②Dlx5, Runx2, Osterix protein expression:Compared with normal group,blank model group increased significantly (P<0.01);compared with blank model group, reinforcing the kidney to replenish marrow traditional Chinese herbs compound group down-regulated significantly(P<0.01),GUSHUKANG granula positive control group also down-regulated significantly(P<0.05),reinforcing the middle to benefit qi granula group and removing blood-house stasis capsule group both tended to decrease,but neither had significant difference statistically(P>0.05).
Conclusions:
1. The method of intramuscular injection of dexamethasone(2.5mg/kg,twice a week,9 weeks continuously) can establish GIOP rat models successfully.The syndrome pathogenesis of GIOP belongs to kidney deficiency and reinforcing the kidney to replenish marrow traditional Chinese herbs compound can prevent and treat GIOP effectively,which has more evident preventative and therapeutic effect on GIOP than the strengthening the spleen herbs and the promoting blood circulation herbs.
2. Normal rats express Runx2,Osterix,Msx2 and Dlx5 at mRNA and protein level in bone tissues and kidney tissues.The kidney storing essence to produce marrow to dominate bones by cascading regulation of Runx2 interrelated genes expression in bone and kidney of kidney system.
3. Long-term and super-physiological dose usage of GC consumes kidney essence, leading to deficiency of kidney essence and bone marrow unable to moisten and nourish bones and kidney,resulting in dysfunction of bones and kidney incapable of regulating Runx2 interrelated genes expression cascadingly.Decrease of Runx2,Osterix,and Dlx5 mRNA and protein expression and increase of Msx2 mRNA and protein expression in bone tissues.Increase of Runx2,Osterix,and Dlx5 mRNA and protein expression and decrease of Msx2 mRNA and protein expression in kidney tissues.Maybe the above-mentioned is one aspect of the microcosmic pathogenesis of GIOP of kidney deficiency.
4. The reinforcing the kidney to replenish marrow traditional Chinese herbs compound with the function of reinforcing the kidney to replenish essence, tonifying marrow to strengthen the bone can recover the kidney’s function of storing essence to produce marrow to dominate bones to regulate Runx2 interrelated genes expression in bone and kidney of kidney system cascadingly.Up-regulating Runx2,Osterix,and Dlx5 mRNA and protein expression and down-regulating Msx2 mRNA and protein expression in bone tissues.Down-regulating Runx2,Osterix,and Dlx5 mRNA and protein expression and up-regulating Msx2 mRNA and protein expression in kidney tissues.Maybe the above-mentioned is one of the mechanisms of reinforcing the kidney to replenish marrow traditional Chinese herbs compound to prevent and treat GIOP.
引文
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