| Object iveThis study aimed to (1) investigate the basic pathogenesis and therapeutic principle of elderly osteoporosis (OP);and (2) analyse the feasibility and rationality of the treatment of elderly OP of kidney deficiency and hepatic stagnation syndrome based on not only analysing the characteristics of traditional Chinese medical (TCM) syndrome but also reviewing literature related to elderly OP under the guidance of traditional Chinese medical theory; and (3) observe the clinical effect of kidney-tonifying and liver-regulating recipes;and (4) explore the mechanism of osteogenic differentiation of aging bone mesenehymal stem cells (BMSCs) induced by the recipes through regulating Wnt signaling pathway according to the theory of aging, homogeny of liver and kidney, and the latest study on the kidneys'physiological functions of storing essence, taking charge of the bone and manufacturing marrow. The assignment served for the purpose of providing new ideas and measures to prevent and cure the eldely OP.MethodsThe systemical research was made on elderly OP through three parts of theory inquiry, clinical observation and cell experiment.1. Theoretical StudiesThe literatures of elderly OP were overviewed, generalized and summarized to analysis the feasibility and rationality of the treatment of elderly OP based on diagnosis of kidney deficiency and hepatic stagnation syndrome.2. Clinical Studies (1) The study of the characteristics of TCM syndrome of elder OPIn this study we chosed the osteoporotic patients as experimental subjects.166cases of out-and in-patients suffering from OP were observed by using dual-functional X-ray bone mineral density apparatus to measure BMD, and judging the syndrome according to the TCM standard. The syndrome database of the patients were established to study the syndrome elements and syndrome combination laws.(2) The clinical study of elderly OP with depressive disorderGeriatric depression scale (GDS) was applyed to assess depressive disorder and analyze the reasons. The syndrome was judged according to the TCM standard. The scores of TCM syndrome and GDS were calculated and analysed to assess the correlativity between TCM syndrome and depressive disorder.(3) The clinical study of the effect of kidney-tonifying and liver-regulating recipes on elderly OP80osteoporotic subjects enrolled in this clinical study were randomly divided into the treatment group and the control group. The clinical symptom improvement and the scores of TCM syndrome were observed, the pain intensities were recorded, and analgesic efficiency was calculated.3. Experimental StudiesBMSCs of SD rats were isolated with adherent separation method and primarily cultured in vitro, morphologically observed under an inverted microscope, and identified by flow cytometry. BMSCs senescence were induced by D-galactose and were detected by β-gal dye staining. The aging BMSCs cultured with conditioned media (Osteo-induced), different concentrations of water extract of kidney-tonifying and liver-regulating recipes and there was nothing as a control. The SAHF of aging BMSCs pretreated with different concentrations of water extract of the recipes were imaged and analyzed by confocal laser scanning microscope.The proliferation of aging BMSCs were detected with MTT assay. The osteogenic differentiation potential of aging BMSCs, which were effected by Chinese herbs, was determined by using standard osteogenic differentiation procedures. Alizarin bordeaux staining method was used to assay the function of cell mineralization. Immunohisto-chemistry and western blot were applied to detect the expression of HIRA, ASFla, Wnt2,β-catenin, GSK33, pGSK3β of the cells. Bands were semi-quantitatively analyzed by Bio-Rad Quantity one Gel-Pro Analyzer software. Results1. Theoretical Studies(1) According to the theory of homogeny of liver and kidney and "kidney controlling bone", kidney deficiency and hepatic stagnation are the key of pathogenesis of elderly OP. Kidney deficiency is the nuclear pathogenesis. Hepatic stagnation is a major disease pathogenesis. Kidney deficiency and hepatic stagnation influence each other and interact as both cause and effect. It is essential to put a great deal of emphasis on both sinews and bones, and to supply the theory homogeny of liver and kidney in the prophylaxis and treatment of elderly osteoporosis. The conclusion establishes a theoretical framework to study the cause and the pathogenesis of POP.(2) The basic therapeutic principle for elderly OP, which attributes to primary deficiency and secondary excess, is to tonify the kidney and regulate the liver. It is extremely important to disperse liver stagnation, fill with liver blood, nourish liver-yin, add up essence to kidney, encourage kidney yang and nourish kidney-yin. This conclusion is the same as the theory framework that we have built.(3) According to TCM theory, the reference of some theories from specialists and by trial and error, we screened Chinese crude drugs to establish kidney-tonifying and liver-regulating recipes, meanwhile, instructed clinical application.2. Clinical Research(1) The main characteristics of syndrome in eldly POP patientsThe high frequency of TCM symptoms of166cases OP patients were back pain, the chest coerces bloated pain, depression, preference for sighing, irritability, blurred vision, limb weakness in pain, fatigue, chills, bitter taste, nocturia increased, tinnitus and deafness, pale or sallow complexion, pale or dark tongue, little and white tongue coating, deep and thin or deep and string pulse.(2) The distribution characteristics of the symptom factor of disease location in eldly POP patientsThe symptom factors of disease location were related to kidney in151cases, liver in140cases, spleen in26cases, heart in10cases, and lung in5cases. The location complex were1iver and kidney in128cases,1iver and spleen in15cases, spleen and kidney in8cases, lung and kidney in5cases, heart and kidney in3cases, heart and spleen in3cases, heart and liver in4cases. The finding suggested that the symptom factor of disease location in eldly POP patients were mainly related to liver and kidney.(3) The distribution characteristics of the syndrome factors of disease nature in eldly POP patientsThe syndrome factors of disease nature mainly included deficiency of qi, qi stagnation, blood stasis, deficiency of Yin, deficiency of Yang. The common syndrome combination nature of eldly POP were tagnation and blood stasis, Yin deficiency and blood stasis, Qi deficiency and blood stasis, Yin and Yang deficiency. The blood stasis was due to qi stagnation commonly. The combination of syndrome factors of disease nature in eldly POP patients mainly include hepatic stagnation and kidney deficiency, Yin deficiency of liver and kidney, Yang deficiency of spleen and kidney.(4) The syndrome combination law of eldly POPThe syndrome combination law of eldly POP was mainly featured as:hepatic stagnation and kidney deficiency.(5) Correlation study among the syndrome factors of disease nature, location and BMD level in eldly POPIt showed no statistically significant difference in the comparison of the syndrome factors of disease nature, location and BMD level(P>0.05). The finding suggested that there were no obvious correlation among the syndrome factors of disease nature, location and BMD level in eldly POP.(6) The incidence of depressive disorder in eldly POP patientsDepressive disorder occurred in81of the osteoporotic cases (48.8per cent), including31cases of mild depression,22cases of moderate depression, and28cases of severe depression.(7) Cause analysis on depressive disorder in elderly POP patientsThere were several major causes of depressive disorder in elderly POP patients. These included body pain, decreased mental and physical endurance, worried about fracture happening again, worried that they could not take care of themselves, increasing the burden to their children, and increasing medical expenses.(8) The correlation analysis between TCM syndrome and depressive disorder in elderly POP patients.Hepatic stagnation and kidney deficiency syndrome was closely related to depressive disorder. Yin deficiency of liver and kidne syndrome tended to depressive disorder easily.(9)The comparison of the clinical efficacy between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndrome32cases insisted on taking kidney-tonifying and liver-regulating recipes, among them,19cases had obvious improvement, and9certain improvement,4cases no response. The total effective rate was87.50%. In control group,31cases insisted on treatment,16cases have obvious improvement, and10certain improvement,5no response. The total effective rate was83.87%. The total effective rate had no significant difference between two groups (P>0.05).(10) The comparison of pain relief between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAmong31cases in the control group,13cases had obvious improvement, and10certain improvement,8no response. The effective rate was74.19%. Among32cases in treatment group,13cases have obvious improvement, and10certain improvement,9no response. The effective rate was71.88%. The effective rate had no significant difference between two groups (P>0.05).(11) The comparison of TCM symptom between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAmong31cases in the control group,11cases were cured, and9obvious improvement,6certain improvement,5no response. The total effective rate was83.87%. Among32cases in treatment group,16cases were cured, and11obvious improvement,3certain improvement,2no response. The total effective rate was93.75%. There was significant difference between two groups (P<0.05).It suggested that kidney-tonifying and liver-regulating recipes could improve TCM symptom more effectively than Fosamax.(12) The comparison of TCM syndrome score between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAfter4weeks of treatment, TCM syndrome scores decreased remarkablely in two groups. There were remarkable differences in TCM syndrome score between at4,3,2weeks post-treatment in two groups (P<0.05).It showed that kidney-tonifying and liver-regulating recipes could improve TCM symptoms more effectively than Fosamax.3. Experimental Studies(1) Isolation, culture, identification of BMSCsThe morphology of cultured BMSCs was consistent with the biological characteristics of BMSCs. The flow cytometry results showed that high expression of CD44was seen with positive rates of96.59(%), and CD2993.86(%), while the low expression of CD34was found with positive rates of0.23%. The results revealed that the cultured BMSCs could meet experimental requirements.(2) BMSCs induced by D-galactose were detected by transmission electron microscopyCompared with control cells, BMSCs induced by D-galactose displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers. There were significant difference between the two groups in cellular structure and morphology.(3) BMSCs senescence induced by D-galactose were detected by β-gal stainingAfter cultured with D-galactose for lldays,23.97±7.89%of the cells were β-gal dye masculine in the normal group, and as were significantly higher in the induced group (p<0.01). After cultured for21days,27.52±10.26%of the cells were β-gal dye masculine in the normal group, while78.81±21.73%in the induced group(p<0.01).But there were no difference between at11days and at21days in the normal group (P>0.05), to the contrary there were significant differencein the induced group(p<0.01).(4) Effect of water extract of kidney-tonifying and liver-regulating recipes on proliferation of aging BMSCsAfter cultured for24h,48h, MTT assay showed that absorbance of BMSCs treated with water extract of kidney-tonifying and liver-regulating recipes at the concentration range of lmg/ml-10mg/ml were significantly higher than that of intervented with no disposal in the blank group (p<0.01). The water extract at the concentration of100mg/ml inhibited the proliferation of BMSCs obviously. However the water extract at the concentration range of0.01mg/ml-0. lmg/ml had no significant effect on the proliferation. There were no difference between the concentration of lmg/mland10mg/ml after24hours (P>0.05). In contrast, the the concentration of10mg/ml increased BMSCs proliferation more significantly than that of lmg/ml after48hours. The above observation demonstrated that the water extract had the effect on promoting proliferation of BMSCs. But it was difficult to reach the concentration of10mg/ml in vivo, thus we concentrated on further confirmation of its effects on BMSCs proliferation at the concentration of lmg/ml.(5) The study of the osteogenic differentiation potential of aging BMSCs induced by water extract of kidney-tonifying and liver-regulating recipesAfter cultured for7d and14d, osteogenic inducer and water extract of kidney-tonifying and liver-regulating recipes promoted formation of mineralization nodes more notebally than the placebo. Significant differences were observed(P<0.01). But after cultured for14d the same were exhibited between the osteo-induced group and the water extract group (P<0.01). A strong increase of formation of mineralization nodes in the water extract groups at all examined time points showed that the water extract had the effect on promoting proliferation of BMSCs.(6) Effect of SAHF formation during BMSCs senescence on different concentration of the water extract of kidney-tonifying and liver-regulating recipes by confocal laser scanning microscopeSAHF formation was universal among BMSCs senescence, while the water extract could reduce SAHF formation. There was significant difference.(7) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the HIRA,ASFla expression of BMSCsImmunohisto-chemistry and western blot measurements revealed that high expression of HIRA,ASFla were observed among aging BMSCs, while low expression among the nomal BMSCs. The water extract reduced HIRA, ASFla expression to postpone BMSCs senescence process.(8) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the Wnt2, β-catenin, GSK3β expression of BMSCsImmunohistochemistry and western blot measurements revealed that expression of Wnt2, β-catenin in the blank group were lower than those in the osteo-induced group and in the water extract group, and the expression of GSK3β in the blank group was higher than that in the osteo-induced group and in the water extract group. It suggested the water extract of kidney-tonifying and liver-regulating recipes and the osteogenic inducer could promote proliferation, differentiation, maturation and mineralization of BMSCs in vitro by increasing the expression of Wnt2,β-catenin and reducing the expression of GSK3β.(9) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the phosphorylated GSK3β protein expression of BMSCsCompared with treated BMSCs, phosphorylated GSK3β protein were reduced in the aging BMSCs, which demonstrated that the water extract of kidney-tonifying and liver-regulating recipes and the osteogenic inducer could increase phosphorylated GSK3β protein.Conclusion1. Kidney deficiency and hepatic stagnation is the main syndrome type of elderly osteoporosis. The method of tonifying the kidney and regulating the liver to treat elderly osteoporosis is logical, rational and clinically practical.2. Kidney deficiency and hepatic stagnation is closely correlated with aging and POP. The pathogenesis of elderly osteoporosis is closely correlated with aging, depression and BMSCs senescence.3. D-galactose can induce BMSCs senescence. The D-galactose induced aging model is a good one for the research of BMSCs senescence.4. Kidney-tonifying and liver-regulating recipes, with their proven efficacy and safety, could be the prospective medicine to treat elderly osteoporosis.5. Kidney-tonifying and liver-regulating recipes treat elderly osteoporosis through reducing SAHF formation via downregulating HIRA, ASFla to postpone BMSCs senescence process.6. The cellular and molecular mechanisms of kidney-tonifying and liver-regulating recipes in treatment of elderly osteoporosis is to promote proliferation, differentiation, maturation and mineralization of BMSCs by upregulating Wnt2,β-catenin expression, downregulating GSK3β expression and increasing phosphorylated GSK3β protein via the activation of Wnt/β-catenin signaling pathway.
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