Study On Substance Basis And Mechanism Of Osteoporosis In Zougui Pills Against Ovariectomized Rats

Purpose:UPLC-Q-TOF-MS technique was employed to conduct the qualitative and relatively quantitative analyses of chemical components which were extracted by 3 methods from Zuogui Pill, a typical prescription for postmenopausal osteoporosis. Pharmacodynamical differences between proliferation rates of BMSCs of PMOP rats were compared. Thus, extraction method was optimized. Pharmacodynamic substance basis was explored. Also, the function mechanism, with which water extracts and alcohol extracts of Zuogui Pill acts on BMSCs osteogenic differentiation of ovariectomized rats, was explored from the aspect of metabolomics and of effects of TGF-β/Smad signaling pathway. Material and Methods:Sixty SPF male nonparous SD rats, 2 months of age, were allowed to acclimate for 7 d, during which water and food were offered ad libitum. 6 PMOP rats model were prepared by bilateral oophorectomy, After 1 week of acclimation, Bilateral femoral and tibial BMSCs were isolated following sacrifice, and then cultured to the 4th generation for experiment. Two best Zuogui Pill extraction methods of the 3 was screened for the following experiments in vivo according to the results of the analysis, in which the three extraction methods were tested employing MTT technique for the effects of intervention in vitro on PMOP rat BMSCs proliferation.The remaining 54 rats were grouped according to the random number table method, 6 animals into Blank Control Group(Control) and 48 animals into Operation Group. PMOP rat model was prepared by bilateral oophorectomy. Animals which survived 3d after bilateral oophorectomy were randomized into 8 groups as follows based on weight stratification: Model Group(OVX), Bujiale Group(BJL), Water Extraction Group High Dosing Sub-Group(GS), Water Extraction Group Middle Dosing Sub-Group(ZS), Water Extraction Group Low Dosing Sub-Group(DS), Alcohol Extraction Group High Dosing Sub-Group(GC), Alcohol Extraction Group Middle Dosing Sub-Group(ZC), Alcohol Extraction Group Low Dosing Sub-Group(DC),6 animal each group. The rats were gavaged once daily for a week, perfusion for 12 weeks, with aliquots of distilled water in Control Group and OVX Group, while with prepared responding dose formula in remaining groups, 6.3 folds of human dosage/kg rat body weight.The serum samples were collected from 15 min, 30 min, 1h and 2h after the post-dose of the high dose group, Chemical components of rat plasma were tested by UPLC-DAD-MS technique. The other rats, blood was collected from abdominal aorta. Metabolomics study on Zuogui Pill for anti-osteoporosis of in ovariectomized rats was conducted with UPLC-QTOF-MS technique. Bilateral femoral and tibial BMSCs were isolated following sacrifice, and then cultured to the 4th generation for experiment. The effects of different doses by 2 extraction methods of Zuogui Pill on rat BMSCs proliferation were tested by MTT technique. Then best doses were screened by PNPP technique in which the activation of BMSCs alkaline phosphatase(ALP) was determined.Fourty SPF male nonparous SD rats, 2 months of age, were allowed to acclimate for 7 d, during which water and food were offered ad libitum. After 1 week of acclimation, the animals above were grouped according to the random number table method, 8 animals into Blank Control Group(Control) and 32 animals into Operation Group. The remaining 32 Normal Group rats were randomized into 4 groups as Model Group(OVX), Bujiale Group(BJL), Zuogui Pill water extraction group(ZGS), and alcohol extraction group(ZGC), 8 animals per group. The rats were gavaged once daily for a week, perfusion for 12 weeks, with aliquots of distilled water in Control Group and OVX Group, while with prepared responding dose formula in remaining groups, 6.3 folds of human dosage/kg rat body weight.The expression of alkaline phosphatase was detected by modified calcium cobalt staining method, and the formation of mineral node was observed by alizarin red staining method. Expression level of BMSCs RUNX2, Collagen I, PPARγ, C/EBPα, and C/EBPβ m RNA in each group were tested by Real Time RT-PCR, respectively. Expression level of BMSCs RUNX2, Collagen I, PPARγ, C/EBPα, C/EBPβ, TGFβ1, TβRⅠ, TβRⅠ, Smad2, Smad3 and Smad4 protein in each group were tested by Western blotting, respectively. Results: 1.Study on the Substance Basis of BMSCs Osteogenic Induction Intervention of Ovariectomized Rats by Different Extraction Method of Zuogui Pill 1.1 Mass Spectrometry Analysis of Compounds in Zuogui Pill Water ExtractsChemical components of medical material, such as prepared rehmannia root(Radix Rehmanniae Preparata), dodder seed(Semen Cuscutae), twotoothed achyranthes root(Radix Achyranthis Bidentatae), barbary wolfberry fruit(Fructus Lycii), common yam rhizome(Rhizoma Dioscoreae), and asiatic cornelian cherry fruit(Fructus Corni), as well as 51 possible compounds, such as Rehmannioside A, dodecane, achyranthoside Ⅰ, achyranthoside Ⅰ, scopoletin, kaempferol, loganic acid, hyperoside, and astragalin, were primarily identified Zuogui Pill water extracts following MS 1 and MS 2 analysis, relative data base research and literature review. 1.2 Mass Spectrometry Analysis of Compounds in Zuogui Pill Alcohol ExtractsChemical components of medical material, such as prepared rehmannia root(Radix Rehmanniae Preparata), dodder seed(Semen Cuscutae), twotoothed achyranthes root(Radix Achyranthis Bidentatae), barbary wolfberry fruit(Fructus Lycii), common yam rhizome(Rhizoma Dioscoreae), and asiatic cornelian cherry fruit(Fructus Corni), as well as 62 possible compounds, such as Rehmannioside A, linoleic acid, aesculetin, oleic acid, dodecane, achyranthoside Ⅰ, achyranthoside Ⅰ, scopoletin, kaempferol, loganic acid,and cor-nuside, were primarily identified in Zuogui Pill alcohol extracts following MS 1 and MS 2 analysis, relative data base research and literature review. 1.3 Mass spectrometry Analysis of Compounds in Water Solution of Zuogui Pill by Water Extraction Combined Alcohol SedimentationChemical components of medical material, such as prepared rehmannia root(Radix Rehmanniae Preparata), dodder seed(Semen Cuscutae), twotoothed achyranthes root(Radix Achyranthis Bidentatae), barbary wolfberry fruit(Fructus Lycii), common yam rhizome(Rhizoma Dioscoreae), and asiatic cornelian cherry fruit(Fructus Corni), as well as 60 possible compounds, such as Rehmannioside A, linoleic acid, aesculetin, oleic acid, dodecane, achyranthoside Ⅰ, achyranthoside Ⅰ, scopoletin, kaempferol, loganic acid,and cor-nuside, were primarily identified in Zuogui Pill water solution of water extraction by alcohol sedimentation following MS 1 and MS 2 analysis, relative data base research and literature review. The components and relative strength of different compounds in the solutions which were extracted from Zuogui Pill by 3 different methods were determined by MS relatively quantitative analysis method. 1.4 BMSCs Flow Cytometry Identification As indicated by the flow cytometry test, CD11b/c(4.9%) and CD45(5.1%) expressed as negative, while CD90(64.5%) and CD29(94.6%) expressed as positive. 1.5 BMSCs Growth CurveDay 1-2 post-inoculation was the incubation phase; Day 3 was the beginning of logarithmic phase of BMSCs proliferation, and Day 4-5 was the peak, then stagnate phase. Thus the growth curve was shaped as an S. 1.6 The Intervention In Vitro of Three Zuogui Pill Extract Types on BMSCs Proliferation of PMOP RatsThe results showed: Three extraction methods and different concentrations of Zuogui Pill had different effect on cell proliferation. All 3 extraction methods had high inhibition of cell at 25mg/ml, and there were no statistically significant difference between groups(P>0.05); There were no statistically significant difference of cell inhibition rate affected by different extraction methods between groups at 2.5mg/ml, 0.025mg/ml, and 0.0025mg/ml(P>0.05); Water extracts had lower inhibition rate than alcohol extracts had at 0.25mg/ml, and compared water decoction with alcohol extraction, cell inhibition rate of alcohol solution of water extraction by alcohol sedimentation group increased significantly than that of water solution of water extraction by alcohol sedimentation group. Water extraction and alcohol extraction had lowest inhibition rate at 0.25mg/ml. 1.7 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs proliferation in PMOP ratsThe results showed: The BMSCs proliferation promotion of PMOP rats which were gavaged with Zuogui Pill water extracts and 70% alcohol extracts were time related. Compared with Control Group on Day 1 and Day 3, proliferation rate of OVX Group decreased significantly(P<0.05); Compared with OVX Group, proliferation rate of BJL, ZS, ZC Group increased significantly(P<0.05); Compared with Control Group on Day 5 and Day 7, proliferation rate of OVX Group decreased significantly(P<0.05); Compared with OVX Group, proliferation rate of BJL, ZS, ZC Group increased significantly(P<0.05), proliferation rate of GS, DS, GC, and DC Group increased significantly(P<0.01), and there was no significant difference between ZS Group, ZC Group and BJL Group(P>0.05). 1.8 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs ALP Activation in PMOP RatsALP Activation of PMOP rat BMSCs was tested by PNPP. ALP activation was highest on Day7 in each group, among which medicine groups had higher ALP activation on Day 7 than on the other days. Compared with Control and OVX Group at 4 time points, ALP activation of water extracts and 70% alcohol extracts of Zuogui Pill had significantly increased in different dose groups and in BJL Group(P<0.05); ALP activation increased most in ZS and ZC Group, among which ZS is higher. 2. Plasma Chemical Components Analysis of Rats Gavaged with Water Extracts of Zuogui Pill and 70% Alcohol ExtractsFor rats gavaged with water extracts of Zuogui Pill, hyperoside and kaempferol were identified in rat plasma sampled 15 min, 30 min, 1 h, and 2h post-dose; For rats gavaged with 70% alcohol extraction of Zuogui Pill, hyperoside and chlorogenic acid were identified in rat plasma sampled 15 min, 30 min, 1 h, and 2h post-dose; 3. Study on Function Mechanism of Anti-Osteoporosis of Zuogui Pill in PMOP Rats: Based on MetabolomicsIndole acetaldehyde, the differential Marker, are interchangeable with tryptophan, which is the precursor of 5-hydroxytryptamine(5-HT). The strength of 5-HT, which is closely related with bone formation and resorption, increased in dose administration groups, which was related to the promotion of bone formation and inhibition of bone resorption.The experimental results showed, The strength of retinyl ester, which is interchangeable with Vitamin A, in administration groups decreased, thus the strength of Vitamin A decreased. As a result, the destruction of bone by Vitamin A was slowed, and the medicine effected.Alpha-CEHC, metabolite of Vitamin E, produces osteoblast, and destructs and absorbs osteoclast, balancing and regulating the metabolism of bone. The experimental results showed: The strength of alpha-CEHC in rats of administration groups decreased, thus the strength of Vitamin E decreased. As a result, the destruction of bone reduced, and the medicine effected. 4 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs Osteogenic Induction and Differentiation in PMOP Rats 4.1 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs ALP Expression in PMOP RatsThe results showed: Stained by modified calcium cobalt staining method, dark brown particle gathered(deposited). Compared with Control Group, the deposit reduction of brown cell was significant in OVX Group; while the density was strengthened in other groups, and the cell became long in shape and more in amount, also, there were more dark brown deposit. 4.2 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs ALP Mineral Nodes in PMOP RatsThe results showed: Obvious mineral nodes were observed and red colored following 14 d of osteogenic induction, 0.1% alizarin red-Tris-HCL and incubation at 37 ℃. In compared with Control Group, nodes in OVX Group reduced obviously and the red was darker, while that in BJL, ZGS, and ZGC Group increased obviously. 4.3 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs RUNX2 m RNA and Protein Expression in PMOP RatsThe results showed: In comparison with Control Group, RUNX2 protein expression was increased significantly in ZGC Group(P<0.05); In comparison with OVX Group, RUNX2 m RNA expression was increased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, RUNX2 m RNA expression was decreased significantly in ZGC Group(P<0.05). RUNX2 m RNA expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05).In comparison with Control Group, RUNX2 protein expression was decreased significantly in other group(P<0.05); In comparison with Group OVX, RUNX2 protein expression was increased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, RUNX2 protein expression was decreased significantly in ZGC Group(P<0.05). RUNX2 protein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.4 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs Col m RⅠNA and Protein Expression in PMOP RatsThe results showed: In comparison with Control Group, Col Ⅰm RNA expression was increased significantly(P<0.05) in OVX Group and ZGC Group, but there was no significant statistical difference for other groups(P>0.05); In comparison with OVX Group, COL Ⅰm RNA expression was increased significantly(P<0.05) in BJL and ZGS Group, but there was no significant statistical difference for ZGC Group(P>0.05); Col Ⅰm RNA expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05).In comparison with Control Group, Col Ⅰprotein expression was decreased significantly(P<0.05) in OVX Group, but there was no significant statistical difference for other groups(P>0.05); In comparison with OVX Group, Col Ⅰprotein expression was increased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, Col Ⅰprotein expression was decreased significantly in ZGC Group(P<0.05). Col Ⅰprotein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.5 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs PPARγ m RNA and Protein Expression in PMOP RatsThe results showed: In comparison with Control Group, PPARγ m RNA expression was increased significantly in OVX, BJL, ZGS, and ZGC Group(P<0.05); In comparison with OVX Group, there was no significant statistical difference for other groups(P>0.05); In comparison with ZGS Group, there was no significant statistical difference in ZGC Group(P>0.05). There was no significant statistical difference between the PPARγ m RNA expressions, at the presence or absence of SB431542(P>0.05).In comparison with Control Group, PPARγ protein expression was increased significantly(P<0.05) in OVX Group, but there was no significant statistical difference for other groups(P>0.05); In comparison with OVX Group, PPARγ protein expression was decreased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, there was no significant statistical difference in ZGC Group(P>0.05). There was no significant statistical difference between the PPARγ protein expressions, at the presence or absence of SB431542(P>0.05). 4.6 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs CEBPα m RNA and Protein Expression in PMOP RatsThe results showed: In comparison with Control Group, C/EBPα m RNA expression was increased significantly(P<0.05) in OVX Group, but there was no significant statistical difference for other groups(P>0.05); In comparison with OVX Group, C/EBPα m RNA expression was decreased significantly(P<0.05) in BJL, and ZGS Group; In comparison with ZGS Group, there was no significant statistical difference in ZGC Group(P>0.05). C/EBPα m RNA expression had been significantly increased since SB431542 was added, in comparison with the absence of SB431542, and the difference was of statistical significance(P<0.05).In comparison with Control Group, C/EBPα protein expression was increased significantly(P<0.05) in OVX, ZGS and ZGC Group, but there was no statistical difference for BJL Group(P>0.05); In comparison with OVX Group, C/EBPα protein expression was decreased significantly(P<0.05) in BJL and ZGS Group; In comparison with ZGS Group, C/EBPα protein expression was increased significantly in ZGC Group(P<0.05). C/EBPα protein expression had been significantly increased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.7 Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs CEBPβ m RNA and Protein Expression in PMOP RatsThe results showed: In comparison with Control Group, C/EBPβ m RNA expression was increased significantly(P<0.05) in OVX Group, but there was no significant statistical difference for other groups(P>0.05); In comparison with OVX Group, C/EBPβ m RNA expression was decreased significantly(P<0.05) in BJL, and ZGS Group; In comparison with ZGS Group, there was no significant statistical difference in ZGC Group(P>0.05). There was no significant statistical difference between the C/EBPβ m RNA expressions, at the presence or absence of SB431542(P>0.05).In comparison with Control Group, C/EBPβ protein expression was increased significantly(P<0.05) in OVX and ZGC Group, but there was no statistical difference for BJL Group or ZGS Group(P>0.05); In comparison with OVX Group, C/EBPβ protein expression was decreased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, C/EBPβ protein expression was increased significantly in ZGC Group(P<0.05). C/EBPβ protein expression had been significantly increased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.8. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs TGF-β1 Protein Expression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, TGF-β1 protein expression was decreased significantly(P<0.05) in OVX Group, BJL and ZGS Group, but there was no statistical difference for ZGC Group(P>0.05); In comparison with OVX Group, TGF-β1 protein expression was increased significantly(P<0.05) in BJL and ZGS Group, but there was no statistical difference for ZGC Group(P>0.05); In comparison with ZGS Group, there was no significant statistical difference for TGF-β1 protein expression in ZGC Group(P>0.05). TGF-β1 protein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.9. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs TβR PrⅠ otein Expression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, TβR Ⅰprotein expression was decreased significantly(P<0.05) in OVX Group and ZGC Group, but there was no significant statistical difference for BJL or ZGS Group(P>0.05); In comparison with OVX Group, TβR Ⅰprotein expression was increased significantly(P<0.05) in BJL and ZGS Group, but there was no statistical difference for ZGC Group(P>0.05); In comparison with ZGS Group, there was no significant statistical difference for TβR Ⅰprotein expression in ZGC Group(P>0.05). TβR Ⅰprotein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.10. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs TβR Protein ⅠExpression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, TβR Ⅰprotein expression was decreased significantly in OVX, BJL, ZGS, and ZGC Group(P<0.05); In comparison with OVX Group, TβR protein expression was increased significantly(Ⅰ P<0.05) in BJL and ZGS Group, but there was no statistical difference for ZGC Group(P>0.05); In comparison with ZGS Group, TβR protein expression was decreased signiⅠ ficantly in ZGC Group(P<0.05). There was no significant statistical difference between the decreases of TβR Ⅰprotein expression, at the presence or absence of SB431542(P>0.05). 4.11. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs Smad2 Protein Expression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, Smad2 protein expression was decreased significantly(P<0.05) in OVX, ZGS and ZGC Group, but there was no statistical difference for BJL Group(P>0.05); In comparison with OVX Group, Smad2 protein expression was increased significantly(P<0.05) in BJL, ZGS, and ZGC Group; In comparison with ZGS Group, Smad2 protein was decreased significantly in ZGC Group, and the difference was statistically significant(P<0.05). Smad2 protein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.12. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs Smad3 Protein Expression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, Smad3 protein expression was decreased significantly in OVX, BJL, ZGS, and ZGC Group(P<0.05); In comparison with OVX Group, Smad3 protein expression was increased significantly(P<0.05) in Group BJL, ZGS, and Group ZGC; In comparison with ZGS Group, Smad3 protein was decreased significantly in ZGC Group, and the difference was statistically significant(P<0.05). Smad3 protein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). 4.13. Effect of Water Extracts and Alcohol Extracts of Zuogui Pill on BMSCs Smad4 Protein Expression by TGF-β1/Smads signaling pathway in PMOP RatsThe results showed: In comparison with Control Group, Smad4 protein expression was decreased significantly in OVX, BJL, ZGS, and ZGC Group(P<0.05); In comparison with OVX Group, Smad4 protein expression was increased significantly(P<0.05) in BJL and ZGS Group, but there was no significant statistical difference for ZGC Group(P>0.05); In comparison with ZGS Group, Smad4 protein was decreased significantly in ZGC Group, and the difference was statistically significant(P<0.05). Smad4 protein expression had been significantly decreased since SB431542 was added, in comparison with the absence of SB431542(P<0.05). Conclusions: 1. There are slight difference between the compounds by the 3 extracting techniques of Zuogui Pill, and the difference in components and strength are related to the efficacy function basis. 2. Both water extracts and alcohol extracts may improve proliferation and ALP activation of BMSCs in PMOP rats; water extract of Zuogui Pill is better than alcohol extracts; Furthermore, water and alcohol extracts can best promote the osteogenic differentiation of BMSCs at middle dose level. 3.Hyperoside and kaempferol, the prototype components in plasma, are identified in the plasma from the rats gavaged with water extracted Zuogui Pill; while hyperoside and chlorogenic acid are identified in the plasma from the rats gavaged with alcohol extracts. 4. The function mechanism of anti-osteoporosis in PMOP rats of Zuogui Pill is associated with tryptophan metabolism and retinoid metabolism pathway, based on the metabolomics studies. 5. Zuogui Pill water extracts and alcohol extracts may perform induction of the osteogenic differentiation, as well as the inhibition of adipogenic differentiation, of BMSCs from PMOP rats by TGF-β1/Smads signaling pathway regulation; water extraction, between the two methods, plays a better role in osteogenic differentiation regulation by signaling pathway intervention.