Studies Of The Shenling Jiangu Capsule's Mchanism Of Anti-Osteoporosis Based On Metabonomics

With the increase of our county's elderly population,the number of people suffering from osteoporosis is gradually increased.It is essential to research drugs in treating osteoporsis and got people's enough attention.Shenling Jiangu capsule is a patent of medicine composition in treating osteoporosis owned by our group.It has the effects of nourishing the liver and kidney ?promoting blood circulation and relieving pain.In this study,we make rats with osteoporosis induced by retinoic acid and make pharmacodynamic studies.We aims to validate its anti-osteoporosis effect.To reveal the mechanism,we try to evaluate it's protective effects on model rat of osteoporosis based on metabolomics metho.To provide a reliable theoretical for the development of new drugsPart one The pharmacodynamic study of Shenling Jiangu Capsule on osteoporosis rats induced by retinoic acid.Objective:We establishe osteoporosis model in rats feding with retinoic acid.We evaluate the effect of Shenling Jiangu capsule on osteoporosis in rats by bone mineral density,biochemical detection indicators,and histopathological observation.And we examine whether the osteoporosis model is successful.Methods:1 Animals and groupsSixty male SD rats(weighting from 300-320 g)were randomly divided into six groups with ten rats per group(Group 1,the blank control group;Group2,the model control group;Group 3,the JG-low group;Group 4,the JG-mid group;Group 5,the JG-high group;Group 6,the positive group.)The blank control group was irrigated with CMC-Na;The model control group was given retinoic acid;Each treatment group and the positive group was administratedof retinoic acid in the morning and given the respective test substance in the afternoon.It last for four consecutive weeks.2 Detection index2.1 BMD: The rats were anesthetized with isoflurane gas.We mesdured the femur BMD at 0 days,7 days,14 days,21 days,28 days using small animal imager.2.2 Sera index:The rats were fasted 24h(water free)before the experi-ment,anesthetized by chloral hydrate.And we took blood in abdominal aortic quickly.The blood was added to the EP tube,centrifuged(3500r/min,10min),separated of serum.And then determined the rat serum's Ca,P,ALP content.2.3 Histopathological detection:Experimental rats were sacrificed after four weeks,and be taken out on the right tibia,immersion fixed in formalin solution.We made the bone decalcificated,then HE stained and observed sections.Results:1 The BMD results:Comparison within each group,BMD before the start of the experiment has no significant difference between the groups(P>0.1).This is consistent with the principle of random assignment.The 7th day had no significant difference in bone mineral density(P>0.1).It shows the drug initially obvious.We count on the 14 th day' BMD.Compared with the normal group,model group and each treatment group were decreased(P <0.05).And compared with the model group,low dose group,high dose group and positive group were significantly increased(P <0.05).Middle dose group also increased,but no significant difference(P>0.1).We count on the 21 th day' BMD.It had no significant difference in bone mineral density(0.05<P<0.1).We count on the21 th day' BMD.Compared with the normal group,the model group is lower significantly(P <0.01).Low-dose group,high dose group and positive group has no significant difference(P> 0.1),medium dose group decreased(0.01 <P<0.05).And compared with model group,high dose group and positive groupwere significantly increased(P <0.01)and low-dose group and middle dose group also had increased(P <0.05).Each group self-comparison,BMD showed dynamic changes.Bone mineral density of control group maintain a certain level,and basically unchanged(P> 0.1).BMD decreased in the model control group with the time prolonged.The bone density was declining.Bone mineral density at 14 days has significant change compared to 0 days(P <0.01).Administered groups' BMD in rats first showed some decline.After administing it has a certain callback.The low-dose group,high dose group and positive group BMD statistic no difference(P >0.1);Changes in bone mineral density in the middle dose group was more apparent.It has significant change at 0th and14 th day(P<0.01),The change reduced at 21 th and 28 th days(P <0.05).2 Biochemical test results:Blood Ca: The calcium's concentration was significantly changed in serum.Model group decreased significantly when compared with the control group(P <0.01).After administrated,the calcium ion concentration has gone up,but still there are differences compared with the control group(P <0.05).It picked up obviously compared with model group.JG-low group and pos group had differences with the model group(P <0.05).JG-mid group and JG-high group had significant differences(P <0.01).Blood P: The phosphorus' s concentration was significantly changed in serum.Model group decreased significantly when compared with the control group(P<0.01).After administrated,the phosphorus ion concentration has gone up.Compared with the control group,JG-low group,JG-mid group and positive group had no difference(P> 0.05).JG-high group increased not obviously.Compared with the model group,JG-mid group significantly increased(P<0.05)and JG-low group was significantly improved(P <0.01).ALP: Compared with the control group,the model group significantly increased(P <0.01).JG-mid group,JG-high group and positive group had no difference(P> 0.05)and JG-low group also slightly increased(P <0.05).Compared with model group,JG-low group had difference(P <0.05)andJG-mid group,JG-high group and positive group had significantly difference(P <0.01).Ca,P,ALP test results show that bone resorption is greater than bone formation induced by retinoic acid.After administration,there are various indicators to control group regression trend.3 The biopsy results:Pathological examination showed that the control group of rats tibia bone marrow cells are densely distributed,no open area,normal cavity size,dense cortical bone and neat edges.Model group had expand marrow cavity,an empty area and severe bone cavity.This shows that the model of osteoporosis is successful.In administration group,the bone cavity is relatively small,and some have a dielectric filled cavity,fuzzy boundaries of cortical bone and marrow cavity.It may be the result of drug's action.Conclusions:Shenling Jiangu capsule has a good anti-osteoporosis effect.It enables to increase bone density,regulate calcium,phosphorus and alkaline phosphatase.It makes them to normal levels.Dense bone marrow cells and bone voids decreases.Part two The metabolomic mechanism study of Shenling Jiangu capsule on osteoporotic rats.Objective:Based on metabonomics,research for the osteoporosis rats model induced by retinoic acid in metabolic characteristics using LC-MS.We aim to looking for differences metabolites.From both the metabolism parameters we evaluated the protective effect of Shenling Jiangu Capsule on rat model of osteoporosis and revealed its mechanism of action.Methods:1 Animals and groups:Same to part one.2 Samples were collected:Plasma samples were collected:The rats were fasted 24h(water free)before the experiment,anesthetizedby chloral hydrate.We collect blood from the abdominal aorta quickly and blood was added to the EP tube with heparin.Centrifuged(3500r / min,10min)and plasma was separated and set in-80?cryopreservation for standby.Tissue samples were collected:We took testis and epididymis in frozen tubes and frozen in liquid nitrogen,-80? cryopreservation for standby after collecting blood.3 Sample preparation:Plasma samples:The blood was reconstituted at 4 ?,scrolled protein precipitation with blood and acetonitrile(1:3)and centrifugated(14000r/min,10min),at 4? low temperature.The supernatant was diluted with water and the sample was vortexed.Tissue samples: We weighed amount testis,and epididymis.The extract acetonitrile and methanol(3: 1)was added,homogenized,mixed,13000r/min,10 min,4 ? for centrifugation.Supernatant was passed through 0.22?m membrane filter to obtain the sample.4 Chromatography and mass spectrometry conditions:System.Date were acquired using AB SCIEX TripleTOF 5600.5 Data Processing:Samples' positive and negative detection mode was measured by Q-Triple 5600 MS.Using AB's markerview 1.2.1 metabolomics analysis software to identify its peak,the peak filter and peak alignment.We finally get a two-dimensional array of data including mass to charge ratio(M/Z)and retention time(Retention time)and peak area.The two-dimensional data matrix obtained above were introduced into the software SIMCA-P for multivariate statistical analysis.6 Identification of differences in metabolite:Using conventional mass spectrometry law,we determine excimer ion peak metabolite by human metabolomics database HMDL for possible metabolites.Results:We processed and analyzed the blood and tissue datas.We identified eightdifferences metabolites on plasma.They are phenylalanine,tryptop-han,cystine,valine,alien acid,mevalonate,creatine phosphate,2-methyl-estrone.We identified nine different metabolites in testis.They're histidin-e,homocysteine cystine,2-phosphoglycerate,mevalonate,purines,ribitol,deox-ycorticosterone and glucose.We identified eight different metabolites in epididymis.They are leucine,histidine,homocysteine,phenylacetyl glycine,lysine,alanyl,purine,3-hydroxybutyric acid and 4-methoxy cinnamic acid.Conclusions:Retinoic acid-induced osteoporosis in rats may be related to in vivo amino acid metabolism,energy metabolism,glucose metabolism,fat metabolism and hormone levels and others.Shenling Jiangu Capsule achieve the anti-osteoporosis effect through multiple channels.