| ObjectiveAfter establishing rat osteoporosis model with spleen and kidney deficiency syndrome, on the basis of OPG/RANKL which is differentiation regulatory factor of osteoclasts and simultaneously selecting VIP/VPAC2which are closely related with Spleen deficiency syndrome and bone metabolism, we would explore the variation characteristics of OPG/RANKL and VIP/VPAC2and the changes of bone resorption and bone formation in the state of spleen deficiency syndrome, kidney deficiency syndrome, and spleen and kidney deficiency syndrome respectively. Moreover, by contrast of the efficacy of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen prescription on rats with spleen and kidney deficiency syndrome and the effect on OPG/RANKL signal pathway and VIP/VPAC2, the mechanism of recuperating kidney and invigorating spleen prescription on osteoporosis may be partly clarified, and experimental evidence for the scientific connotation of "spleen and kidney related" will be further provided.MethodsFirst, one hundred and ten female Wistar rats were selected and divided into four groups randomly:twelve in normal control group, spleen deficiency model group, sham operation group; seventy-four in bilateral ovariectomized group. After the surgery for10weeks, these seventy-four bilateral ovariectomized rats were divided into six sub-groups again:twelve in kidney deficiency model group, spleen and kidney deficiency model group, diethylstilbestrol group and invigorating spleen group; thirteen in recuperating kidney group and recuperating kidney and invigorating spleen group.Kidney deficiency syndrome model was Established by Ovariectomy after anesthetized by intraperitoneal injection of sodium pentobarbital, while only the fatty tissue near the ovaries of the rats were extirpated in sham operation group;200% rhubarb concentrated decoction was given orally every day for two weeks, then changed to2times a week for three months to establish the spleen deficiency syndrome model; on this basis, the rat osteoporosis model with spleen and kidney deficiency syndrome has been established by the above two methods after Ovariectomy for ten weeks.Diethylstilbestrol group was administrated diethylstilbestrol orally which was diluted to0.0048mg/ml with normal saline; invigorating spleen group, recuperating kidney group, recuperating kidney and invigorating spleen group were given orally Sijunzitang, Guiluerxiantang, and the two combined respectively, The drug or decoctions were given once a day, six days continuously then1day pause, the whole period lasted3months. Distilled water was administrated orally to other groups, at the same time. The rats of all groups were weighed once a week, and adjusted the dose accordingly.At the third day and the sixteenth day before the day all the rats were killed, hydrochloric acheomycin was injected into the abdomen of the rats respectively according to30mg/kg to mark the bone with fluorescence. After Medication, The rats were anesthetized to get artery blood, then the blood was centrifuged to detect the level of Various cytokines in serum by enzyme-linked immunosorbent assay (ELISA).After the rats being killed, the left femur were taken for bone mineral density testing; the right tibia of the rats were retained for bone tissue section which weren’t decalcified to evaluate the change of bone histomorphometry; at the same time the left tibia of the rats were used for frozen tissue section after being decalcified to observe the protein and mRNA expression of VPAC2,OPG and RANKL by means of immunohistochemistry and in situ hybridization; in addition,colon tissue were taken for paraffin tissue section to observe the protein and mRNA expression of VIP by means of immunohistochemistry and in situ hybridization.Results were presented as Mean±SE, normal distribution and homogeneity of variance of the data were analyzed by one-way ANOVA, between any two groups, LSD test; heterogeneity of variance of the data were analyzed by Dunnett’s T3test. And non-normal data by non-parametric tests.Results1Effects of recuperating kidney and invigorating spleen prescription on femur BMD of osteoporosis rats of spleen and kidney deficiency syndrome modelCompared with sham operation group, femur BMD in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, and the latter was more significantly. But Spleen model group was not different.Compared with spleen and kidney deficiency syndrome model group, femur BMD in all treatment groups increased obviously. In recuperating kidney group and recuperating kidney and invigorating spleen group, femur BMD increased more significantly compared with invigorating spleen group, but there was no difference between them.2Effects of recuperating kidney and invigorating spleen prescription on the bone histomorphometry indicators of osteoporosis rats of spleen and kidney deficiency syndrome modelCompared with sham operation group, TBV%in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, TRS%, TFS%, OSW, MAR and mAR increased significantly, while Spleen model group was not significantly different. Compared with kidney deficiency model group, TBV%in spleen and kidney deficiency model group decreased obviously, TRS%and TFS%increased significantly.Compared with spleen and kidney deficiency syndrome model group, TBV%in all treatment groups increased obviously, TRS%、 TFS%、OSW、 MAR and mAR decreased obviously. Compared with invigorating spleen group, TBV%increased and TRS%、 TFS%decreased obviously in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter reversed these indicators more significantly.3Effects of recuperating kidney and invigorating spleen prescription on serum level of OPQ RANKL, VIP, MTL and GASCompared with sham operation group, the serum level of OPG, MTL and GAS decreased significantly in three model groups, except that the serum level of OPG in spleen deficiency model group was not obviously different. In addition, Spleen and kidney deficiency model group was the lowest. While the serum level of RANKL and VIP increased significantly in three model groups, moreover, Spleen and kidney deficiency model group was the highest.Compared with spleen and kidney deficiency syndrome model group, the serum level of OPG increased significantly in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter increased more obviously; the serum level of MTL and GAS increased significantly in invigorating spleen group and recuperating kidney and invigorating spleen group. While the serum level of RANKL decreased significantly in all treatment groups, and among which diethylstilbestrol group and recuperating kidney and invigorating spleen group were optimal; the serum level of VIP decreased significantly in all treatment groups except for diethylstilbestrol group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased more significantly than recuperating kidney group.4Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of OPG, RANKL and VPAC2Compared with sham operation group, the protein and mRNA expression of OPG was not obviously different in spleen deficiency model group and decreased significantly in kidney deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly lower than the former. Different from OPG, the protein and mRNA expression of RANKL increased significantly in all model groups, and kidney deficiency model group was significantly higher than spleen deficiency model group, spleen and kidney deficiency group was higher than kidney deficiency group. In respect of VPAC2, the protein and mRNA expression in three model groups increased significantly compared with sham operation group, compared with kidney deficiency model group, spleen deficiency model group, spleen and kidney deficiency group increased more, and there was no obvious difference between the two groups. Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of OPG increased obviously in all treatment groups, but still lower than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group increased more and no obvious difference between them. In contrast, the protein and mRNA expression of RANKL decreased obviously in all treatment groups, but still higher than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group decreased obviously, and the latter decreased more significantly in mRNA expression. In respect of VPAC2, the protein and mRNA expression decreased obviously in all treatment groups except for diethylstilbestrol group. Compared with recuperating kidney group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased obviously, and the former decreased more significantly.5Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of VIP in colon tissueCompared with sham operation group, the protein and mRNA expression of VIP in colon tissue was not obviously different in kidney deficiency model group and increased significantly in spleen deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly higher than the former.Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of VIP in colon tissue decreased obviously in invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group, but was not different in diethyl stilbestrol group. The protein and mRNA expression of invigorating spleen group and recuperating kidney and invigorating spleen group decreased significantly compared with recuperating kidney group, and there was no difference between them.Conclusion(1) Ovariectomy can cause rat osteoporosis with kidney deficiency syndrome, simply giving the rhubarb concentrated decoction can cause rat spleen deficiency syndrome, but can not lead to osteoporosis. If the two methods combined, which can lead to rat osteoporosis with spleen and kidney deficiency syndrome, and the disease is more serious than that which led by Simple ovariectomy. This shows that both spleen and kidney have important role in the process of osteoporosis, and among which, kidney is fundamental, spleen is one of the important factors.(2) The prescription of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen all have therapeutic effects on osteoporosis rats with spleen and kidney deficiency syndrome, and that of recuperating kidney and invigorating spleen works best, followed by recuperating kidney, and followed by invigorating spleen.(3) The common pathway of three prescriptions is:On the one hand, it can directly regulate the expression of OPG and RANKL, the expression of OPG up-regulated and RANKL down-regulated, so that the osteoclasts activity decreased obviously; on the other hand, it can down-regulate the expression of VIP and VPAC2and thereby reduce the combination of VIP and VPAC2, which can up-regulate OPG levels and down-regulate RANKL levels probably by osteoblast-mediated effect, and ultimately led to reduction of the osteoclasts activity and the bone resorption. In addition, MTL and GAS levels can also be raised significantly in peripheral blood. However, there are some differences among the three ways:invigorating spleen prescription played a stronger role in MTL, GAS, VIP and VPAC2; although the effects of recuperating kidney Prescription on these indicators were weaker than the former, but it can play more significant role in OPG and RANKL; while these indicators were improved significantly by recuperating kidney and invigorating spleen prescription. This may be one of the mechanisms of recuperating kidney and invigorating spleen prescription which was more effective than the other two prescriptions, it further demonstrates that spleen and kidney in Traditional Chinese Medicine are closely related in the occurrence and treatment process of osteoporosis. |
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