| 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. |
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