| Part one Clinical Study of Qianggu Capsule to treat patients with Senile OsteoporosisBackground:Clinically,senile osteoporosis is a common disease.In recent years,the incidence of senile osteoporosis has dramatically increased.As one of osteoporotic complications,fragility fracture threatens the health of the elder,resulting in high mortality and morbidity.Due to the complexity of its pathogenesis,it is recommended that anti-osteoporosis therapy should be based on the lifestyle intervention,the decreased osteogenic function is believed to be the main cause of senile osteoporosis,but bisphosphonates are the first-line treatment of osteoporosis via the inhibition of bone resorption.And unfortunately,does not improve osteogenic dysfunction.Recently,the association of vascular disease and osteoporosis draw more attention.Arteriosclerosis and osteoporosis coexist and their interaction aggravate the pathogenetic condition.It is supposed that bone ischemia caused by arteriosclerosis may be an important factor for senile osteoporosis.Vascular endothelial growth factor(VEGF),a key factor associated with vascular and bone metabolism,significantly reduces in osteoporosis and promotes osteogenic differentiation of BMSCs after activation.Traditional Chinese medicine has a few advantages for osteoporosis.Drynaria fortunei is proved to promote the formation of bone,and consistent with the pathogenesis of the atrophic debility of bones.Its,Rhizoma drynariae is the main active ingredient of drynaria fortunei,but the action and mechanisms remains unclear.Objectives:To clarify the clinical efficacy of Qianggu Capsule(Total Flavonoids of Rhizoma Drynariae)in the treatment of senile osteoporosis,as well as the effects on vascular endothelial growth factor,bone turnover index,bone mineral density and risk of fallin,and to explore its possible mechanism.Methods:Sixty four elderly patients with osteoporosis who met the inclusion criteria were randomly divided into two groups,including the Qianggu capsule group(n=32)and the control group(n=32).Calcium and vitamin D supplements were given to the two groups.The control group did not receive any treatments.The Qianggu capsule group was given one Qianggu capsule,3 times a day after meal.Totally,the duration is 24 weeks.The changes of TCM syndrome scores,blood calcium,phosphorus,alkaline phosphatase,bone biochemical markers,25-hydroxyvitamin D,parathyroid hormone,vascular endothelial growth factor,and bone mineral density,risk of falling were measured before and after the treatment for all the patients.Results:A total of 64 SOP patients were included in the study,including 22 in men and 42 in women,with an average age of 75.77±4.25 years.Which 40 patients with peripheral arteriosclerosis(by ABI index),arter:iosclerosis Group(AS group)had lower bone density than non-arteriosclerosis group(NAS Group),(The AS group had a lumbar vertebrae density of 0.7610.01,a femoral neck bone density of 0.57±0.01,and a total hip bone density of 0.61±0.01.The NAS group had a lumbar spine bone density of 0.85±0.01,a femoral neck bone density of 0.63±0.01,and a total hip bone density of 0.64±0.01).The decrease of osteogenesis function was more obvious,the type 1 procollagen amino terminal peptide(PINP)was 33.75±7.47ng/mL,and the alkaline phosphatase(ALP)was 68.63±14.32mmol/L,which was lower than that of the NAS group(P1NP 42.72±8.58ng/mL,ALP 73.95±12.19mmol/L),and lower VEGF levels(ng/L)(AS group 75.17± 14.32,NAS group 91.35±15.29)ng/L.1.At the end of the intervention,2 patient(N=30)fell from the Qianggu capsule group and 1 patients(n=31)fell from the control group.There were no differences in TCM syndrome scores between the two groups before treatment(28.47±11.43 vs 27.87±11.51),and they all decreased in both groups after treatment(18.66±4.5 vs 26.32±9.29),the reduction of scores in Qianggu capsule group was more significant than that in control group(p<0.01).The clinical symptoms of patients such as lumbar muscle pain,muscular soreness,myasthenia,and trudge improved than before treatment,VAS score has also been significantly improved,the effective rate of treatment reached 80%.The total effective rate of the Qianggu capsule group was significantly higher than that of the control group(p<0.01).2.The biomarkers such as P1NP,β-CTX,25-OHD and ALP were similar in both groups at baseline.A significant increase of P1NP in the Qianggu capsule group was observed after treatment(37.28±10.41 vs 41.02±10.29)(p<0.05),while it was unchanged in control group(37.10±11.58 vs 38.67±10.18).And there was also a statistically significant differnence between two groups.β-CTX in both groups decreased compared with baseline,but the change was not significant within group or between groups.After treatment,the average 25-OHD level was significantly higher than baseline,p<0.05,but the changes were not obvious between the two groups.3.After treatment,there was no statistically significant difference in bone mineral density between the two groups.4.The level of VEGF in the Qianggu capsule group was significantly increased after treatment(84.74±17.98,95.34±18.03)ng/L.In the control group,The VEGF level were(85.75±19.64,87.36±15.63)ng/L before and after treatment,appears a significant elevation within group or between groups(P<0.01).5.The risk assessment of fall showed that daily activity status in Qianggu capsule group were significant improved after treatment 1.13±0.52,0.73±0.27,P<0.05),and balance ability has also been improved(1.03±0.17 vs 1.47±0.39,P<0.01),Compared with the control group,but there are no difference between the two groups.Conclusions:1.the patients with arter:iosclerosis had lower bone density,VEGF levels.2.Qianggu Capsule can significantly improve the TCM syndrome scores,osteogenesis and promote the secretion of VEGF,maintain bone density,reduce the risk of falls with senile osteoporosis.3.Qianggu Capsule may improve osteogenesis bia stimulating the secretion of VEGF.Part two The Mechanism of Qianggu Capsule regulating VEGF biomagnification Coefficient on osteoblast function in rats with peripheral ischemic lesionBackground:Our clinical research shows that Qianggu Capsule is effective on the treatment of senile osteoporosis,which can increase VEGF level,improve P1NP and maintain bone density,but the mechanism still remains unclear.It is currently believed that inadequate blood supply involved the etiology of senile osteoporosis.VEGF is a key regulator of vasculopathy and osteogenesis,which promotes osteogenesis via activating BMSCs,whereas the Wnt/β-catenin signaling pathway is an essential pathway for bone cell transformation.Objectives:To observe the effect of Qianggu Capsule on osteoblast function in elder rats with peripheral arterial ischemia and the role of osteogenic differentiation via regulating VEGF.Methods:48 SPF female SD rats,18 months old,were randomly divided into 2 groups,including the sham operation group(N=5)and the model group(N=43).The model group was given left femoral artery ligation,and femoral artery color Doppler ultrasound was used to observe arterial blood flow the next day.The blood flow blockade was seen as a successful model.Totally,35 model rats were enrolled in the follow-up experment.Rats in the model group were randomly divided into 3 groups,including the control group(N=10),the low-dose group of Qianggu Capsule(N=10),and the high-dose group of Qianggu Capsule(N=15).The 3rd day of Ligation began to intervene,the low-dose group and the high-dose group of Qianggu Capsule were given Qianggu Capsule everyday,and the sham operation group and the model group were given normal saline.The duration of the three groups are 4 weeks.The VEGF were measured on 2nd,2 weeks and 4 weeks after ligation,At the end of the experiment,blood was drawn,and serum was centrifuged to detect bone turnover markers,VEGF and calcium and phosphorus indicators.Femur bone density was measured.Left femur HE staining and CD34 labeled blood vessels were used to observe the local pathological changes of the femur.PCR was used to detect the mRNA expression of VEGF and OPQ Western blot was used to detect the changes of VEGF and RUNX2 of bone tissue.Results:The next day after femoral artery ligation,ultrasound imaging showed that blood flow was blocked,indicating the model successfully established.Molding rate is 81.4%.After 4 weeks of femoral artery ligation,the left femur density of the model group was significantly lower than that of the sham operation Group,suggesting that ischemia of peripheral artery could cause bone mineral density to decrease and induce osteoporosis.1.On 2nd,the levels of VEGF in the model group,the low dose group and the high dose group were significantly higher,which was more statistically significant than that of the sham Operation Group(P<0.01),but there was no significant difference between the 3 groups(P>0.05).2 weeks after ligation,the model group was lower than that of sham Operation Group,There were statistical differences(P<0.05),the low dose group and the high dose group began to fall back from the 2nd day after operation,there were statistical differences(P<0.01),but they were significantly higher than the model group(P<0.01),and there was no significant difference between the low dose group and the high dose group in the 2 weeks after Operation(P>0.05).At 4 weeks after operation,the level of VEGF in the model group continued to decrease,compared with 2nd,2 weeks after operation,there were clear statistical differences(P<0.01),low dose group and high dose group VEGF level is still higher than the same period model group,the difference is statistically significant(P<0.01).The low dose group began to fall back 4 weeks from 2 weeks,the difference was statistically significant(P<0.01),the high dose group was not statistically different from 2 weeks(P>0.05),but there was a clear statistical difference(P<0.01)compared with the lower dose group in the same period.2.There was no difference in the calcium and phosphorus levels between the model group and the sham operation group,but after the administration of high-dose drug,the blood calcium level increased significantly(P<0.01).The BALP and BGP of the model group were significantly lower than that of the sham operation group,P<0.01.The BALP and BGP of the low dose group and high dose groups were significantly increased after the intervention(P<0.01).The level of β-CTX elevated in the model group,while no significant difference was observed.After treatment,a significant decrease in β-CTX was observed in the high-dose group,P<0.01.3.The bone mineral density of the left femur in the model group was significantly lower than that of the sham operation group,P<0.05.After the administration of Qianggu capsule,the bone density increased in a dose-dependent manner,P<0.05.There was no significant difference between the right side of the model group and the sham operation group,but after the treatment of Qianggu Capsule,The bone mineral density of the right femur in the high dose group was significantly increased,P<0.01.4.For the pathology,it can be observed that after the ligation,the femoral vascular density of the model group is decreased,and the trabecular bone is thinned.After the treatment,the blood vessel density and the trabecular bone width increases.5.The VEGF and OPG genes in the model group were significantly lower than that of the sham operation group(P<0.01).Compared with the model group,the high-dose group significantly increased the mRNA levels of VEGF and OPG(P<0.01).Compared with the model group,the mRNA levels of VEGF and OPG were also increased in the low-dose group,and the mRNA level of OPG was significantly increased(P<0.01),but the effect was not as well as that in the high-dose group,suggesting that the effect was dose-dependent.6.Compared with the sham operation group,the protein expression levels of VEGF and RUNX2 in the femur of the model group were significantly lower than those in the sham operation group(P<0.01).Compared with the model group,both high-dose and low-dose groups significantly increased the protein expression levels of VEGF and RUNX2(P<0.05)in a dose-dependent manner.Conclusions:1.Qianggu Capsule can effectively increase vascular density,VEGF level,and promote osteogenesis and increase bone density.2.Qianggu Capsule can activate BMSCs of osteogenic differentiation through VEGF,and play a protective role in blood vessels and bones. |
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