| Purpose:To explore the relevance between "Kidney" and "Bone" in Chinese medicine,this study research the effects of Zuogui pill, Yougui pill and their disassembled prescriptions on relevant index of osteogenic differentiation and Fatty differentiation in ovariectomized rats' kidney through TGF-?1/Smads signaling pathway. All those practice have been made on basis of the theory called “the kidney dominating bones”.Material and method:Dividing 90 SPF female rats at 2 months into 3 groups randomly: Normal group with 10 rats, Sham group with 10 rats, for the rest 70 rats were ovariectomized model group.Ovariectomized model group established by bilateraldorsal ovary resection, an-d sham group only eccoped the fat tissue around the ovaries. After 7 days, dividing the survived rats into 9groups randomly: Control group, Sham group, OVX group, BJL group, ZGW group, YGW group, GTY group, ZYY group, BYY group. Each of treatment group was given corresponding drugs, control group,sham group and modelgroup were administered with distilled water. After 12 weeks drug treatment,all the rats were drawing materials, excluding the death caused by the mold gavage,including 76 rats. 24 hours before operation the rats were no eating but giving water, 2 h after the last administration weighed and gave anesthesia,removal from ratfemur, which was wrapped in wet gauze placed-80 ? frozen for dual-energy X-ray absorptiometry detection; taking out of one third the distal femur and kidney in rats, carefully peeling adhesion of muscle fascia and connective tissue, making paraffin section after the fixed or decalcified for few weeks, in order to prepare for morphology HE detection; The body weight was measured by electronic balance before anesthesia, taking out of the kidney in rats, carefully peeling adhesion of muscle fascia and connective tissue,use torsion balance to measure the wet weight of the kidney for kidney index; taking out of the kidney in rats, carefully peeling adhesion of muscle fascia and connective tissue,after frozen in liquid which was wrapped by nitrogenthe foil, and stored in-80 ? frozen for real-time quantitative PCR was used to detect kidney tissue gene expression Runx2,Collagen?,ALP,BGP,PPAR?,FABP4,TGF-?1,T?R?,T?R?,Smad1,Smad2,Smad4 and Western blot assay of kidney tissue the protein expression of Runx2, Collagen?, ALP,BGP, PPAR?, FABP4,TGF-?1,T?R?,T?R?,Smad1,Smad2,Smad4; taking out of kidney in rats, carefully peeling adhesion of muscle fascia and connective tissue, making paraffin section after the fixed for few weeks, in order to prepare for Runx2, Collagen?, ALP, BGP,PPAR?, FABP4 indicators for immunohistochemistry.Results:1. The impact for ovariectomized rats on bone mineral density for Zuogui pill, Yougui pill and their disassembled prescriptionsCompared with the Control group, except for the Sham group, BMD in each group has a downward trend, rat total femoral BMD was significantly decreased in OVX group(P?0.01);after 12 weeks,compared with OVX group, each treatment group for bone density was improved indifferent degrees(P?0.05 or P?0.01), which the effects of BJL group and ZGW group were better,although YGW group has a significant difference(P ? 0.01), but the level was lower. Comparision results between the other groups were no significant difference.2. The morphological effects on bone tissue ovariectomized rats for Zuogui pill, Yougui pill and their disassembled prescriptionsThe results showed that, Control group and the Sham group showed trabecular bone tightly packed, which morphological structure was complete and trabecular bone was connected by reticular, trabecular bone surface was more visible and a single row of osteoblasts, marrow cavity was relatively small. While the total number of trabecular bone in the OVX group was significantly reduced, the structure was more sparse, connectivity was poor and irregular, showing a large number of blind side in trabecular bone, trabecular thickness of the wall was inconsistency, a number of bone cells were turned into trabecular bone surface, which was significantly decreased, and seen bulky, irregular and multinucleated osteoclasts, and the bone marrow cavity increased. Each treatment group after treatment for12 weeks, the number of trabecular bone have been increased and widened in different degrees, structural arrangement and shape gradually approaching the OVX group, and it was shown that trabecular bone was characterized by reticular connection and marrow cavity which was narrowing. In all groups, effects of BJL group, ZGW group were marked.3. The kindey index effects on kidney tissue ovariectomized rats for Zuogui pill, Yougui pill and their disassembled prescriptionsCompared with Control group, The kidney index of OVX group js lower(P?0.01), eachtreatment group for kindey index was improved indifferent degrees, the difference was statistically significant(P ? 0.05); compared with OVX group, the kidney index of each treatment group were improved, the left kidney index of ZGW and YGW were improved obvious(P?0.05), the right kidney index and total kidney index of ZGW?YGW?ZYY were statistically significant(P ? 0.01); compared with BJL group, the kidney index of each treatment group were lower than BJL group,and GTY group and BYY group have statistically significant(P?0.05).4. The morphological effects on kindey tissue ovariectomized rats for Zuogui pill, Yougui pill and their disassembled prescriptionsThe results showed that,in control group and sham group, glomerulus was closely linked with visceral layer of fowmans capsule,the structure was clear and complete, the structure of proximal convoluted tubule was clear; renal corpuscles of OVX group structure disordered,glomerular and renal capsule cannot distinguish, tubular structure of renal body was destroyed;in BJL group,ZGWgroup,YGW group,ZYY group could discovered clear and complete renal corpuscles.5. The impact on ovariectomized rats' kidney Runx2?Collagen??ALP?BGP for Zuogui pill,Yougui pill and their disassembled prescriptionsImmunohistochemical detection method results show that: in control group and sham group, glomerulus was closely linked with visceral layer of fowmans capsule,the structure was clear and complete, the structure of proximal convoluted tubule was clear. It was seen dark nankeen positive expression of ALP and BGP in tubular section,and the nucleus have no obvious positive expression.In glomerular and tubular interstitial, could seen dark nankeen positive expression of Runx2 ? Collagen ?; renal corpuscles of OVX group structure disordered, glomerular and renal capsule cannot distinguish, tubular structure of renal body was destroyed, It could not seen positive expression of ALP and BGP in tubular section cells,there is no dyeing in distal tubules.The expression of Runx2?Collagen?in glomerular and tubular interstitial were not obvious; renal corpuscles' structure in BJL,ZGW,YGW,ZYY were clear and complete, GTY,BYY were a little weaker than them, the number of ALP,BGPpositively staining cells and Runx2 ? Collagen ? positively staining cells were increased in some extend, which ZGW group in treatment group is better.Gene expression results: Compared with the Control group, expression of Runx2,Collagen?, ALP, BGP in OVX group were decreased, there was a significant difference(P?0.01); compared with the OVX group, the gene expression of Runx2, Collagen?, ALP,BGP in BJL group,ZGW group,YGW group were increased in different degrees(P ? 0.01);compared with the BJL group, the gene expression of each treatment group Runx2, Collagen?, ALP, BGP reduced in different degrees(P ? 0.01), while the content in ZGW group increased significantly.Protein expression results: Compared with the Control group, expression of Runx2,Collagen?, ALP, BGP in OVX group were decreased, there was a significant difference(P? 0.01); after 12 weeks, the treatment group Runx2, Collagen ?, ALP, BGP protein expression were increased in different degrees(P?0.01); compared with the BJL group, the expression of each treatment group Runx2, Collagen ?, ALP, BGP reduced in different degrees(P?0.01), while the content in ZGW group increased significantly.6. The impact on ovariectomized rats' kidney PPAR?,FABP4 for Zuogui pill, Yougui pill and their disassembled prescriptionsImmunohistochemical detection method results show that:PPAR?: in control group and sham group, glomerulus was closely linked with visceral layer of fowmans capsule,the structure was clear and complete, the structure of proximal convoluted tubule was clear.No dark nankeen positive expression were seen; renal corpuscles of OVX group structure disordered, glomerular and renal capsule cannot distinguish, tubular structure of renal body was destroyed, high level expression were seen in Inner medullary collecting duct; renal corpuscles' structure in BJL,ZGW,YGW,ZYY were clear and complete, BYY were a little weaker than them, the number of positively staining cells were increased in some extend.FABP4: To observe the renal medulla and the proximal medullary cortex, in control group and sham group, glomerulus was closely linked with visceral layer of fowmans capsule,the structure was clear and complete, the structure of proximal convoluted tubule wasclear. No dark nankeen positive expression were seen in the cytoplasm and nucles of endothelial cells; renal corpuscles of OVX group structure disordered, glomerular and renal capsule cannot distinguish, tubular structure of renal body was destroyed, high level expression were seen in cytoplasm and nucles of endothelial cells; renal corpuscles' structure in BJL,ZGW,YGW,ZYY were clear and complete, GTY and BYY were a little weaker than them, the number of positively staining cells were increased in some extend.Gene expression results: Compared with the Control group, expression of PPAR? and FABP4 in OVX group were increased, there was a significant difference(P?0.01); compared with the OVX group, the gene expression of PPAR? and FABP4 in each group were decreased in different degrees(P?0.01); compared with the BJL group, the gene expression of each treatment group PPAR? and FABP4 increased in different degrees(P ? 0.01), while the content in ZGW group lower significantly.Protein expression results: Compared with the Control group, expression of PPAR? and FABP4 in OVX group were increased, there was a significant difference(P?0.01); after 12 weeks, the treatment group PPAR? and FABP4 protein expression were decreased in different degrees(P ? 0.01); compared with the BJL group, the expression of each treatment group PPAR? and FABP4 higher than BJL group(P ? 0.01), while the content in ZGW group decreased significantly.7. The impact on ovariectomized rats' kidney TGF-?1/Smads singnal pathways for Zuogui pill,Yougui pill and their disassembled prescriptionsGene expression results: Compared with the Control group, expression of TGF-?1 ?T?R?? T?R?? Smad1 ? Smad2 ? Smad4 in OVX group were decreased, there was a significant difference(P ? 0.01); compared with the OVX group, the gene expression of TGF-?1?T?R??T?R??Smad1 ?Smad2?Smad4 in eavh group were increased in different degrees(P ? 0.01); compared with the BJL group, the gene expression of each treatment group TGF-?1?T?R??T?R??Smad1 ?Smad2?Smad4 reduced in different degrees(P?0.01), while the content in ZGW group increased significantly.Protein expression results: Compared with the Control group, expression of TGF-?1 ?T?R?? T?R?? Smad1 ? Smad2 ? Smad4 in OVX group were decreased, there was a significant difference(P ? 0.01); after 12 weeks, the treatment group TGF-?1 ? T?R? ?T?R??Smad1 ?Smad2?Smad4 protein expression were increased in different degrees(P?0.01); compared with the BJL group, the expression of each treatment group TGF-?1?T?R??T?R??Smad1 ?Smad2?Smad4 reduced in different degrees(P?0.01), while the content in ZGW group increased significantly.Conclusion:1. After eccoping the both ovaries of rats, bone density decreased,microarchitectural deterioration of bone tissue, the PMOP models were duplicate successfully; the kidney index of the experimental rats was decreased in different degrees, morphology of kidneys were changed, it indicates that the occurrence of osteoporosis in postmenopausal women may have a certain effect on the kidney.2. After the ovary, the gene and protein expression of Runx2, collagen I, ALP, BGP in kidney were decreased, These parameters are related to osteogenic differentiation. The changes of bone markers in kidney can be used as a reference index for the evaluation of bone metabolism.3. After the ovary, the gene and protein expression of PPAR??FABP4 were increased, It is suggested that the incidence of osteoporosis in postmenopausal women may have an effect on the lipid metabolism in the kidney.4. Zuogui pill and Yougui pill and its disassembled prescriptions can be adjusted to the kidney of ovariectomized rats in osteogenic and adipogenic differentiation related indicators.5. The occurrence mechanism of osteoporosis in postmenopausal women is related to the imbalance of bone formation and fat balance, Zuogui pill and Yougui pill and its disassembled prescriptions can inhibit the kidney adipogenic differentiation index expression,and promote the expression of osteogenesis related indicators, It's indicates that Zuogui pill and Yougui pill and its disassembled prescriptions in the prevention and treatment of postmenopausal osteoporosis mechanism might be related to the regulation of kidney into osteogenic differentiation and adipogenic differentiation r balance. Among them, the effect ismore prominent in the Zuogui pill group.6. TGF?-1/Smads signaling pathway is correlated with osteogenic differentiation and fatty differentiation. Zuogui pill and Yougui pill and its disassembled prescriptions can inhibit the kidney adipogenic differentiation index expression, nd promote the expression of osteogenesis related indicators is realized through the regulation of TGF?-1/Smads signaling pathway. This provides a certain experimental basis for the correlation between "kidney" and "bone" based on the theory of " the kidney dominating bones " in Chinese medicine. |
PDF Full Text Request