OBJECTIVE: To explore the multi-component synergistic mechanism of Zuogui Wan(左归丸, ZGW) in treating postmenopausal osteoporosis(PMOP). METHODS: The main components and target genes of ZGW were screened via the Trad...OBJECTIVE: To explore the multi-component synergistic mechanism of Zuogui Wan(左归丸, ZGW) in treating postmenopausal osteoporosis(PMOP). METHODS: The main components and target genes of ZGW were screened via the Traditional Chinese Medicine Systems Pharmacology(TCMSP). In addition, the target gene sets of PMOP were derived from the Gene Cards and Online Mendelian Inheritance in Man databases. The search tool for recurring instances of neighbouring genes(STRING) 11.0 software was used to analyze the interaction among intersecting genes. Cytoscape 3.6.1 software and the Matthews correlation coefficient(MCC) algorithm were used to screen the core genes. Fifty Sprague-Dawley female rats were randomly divided into the sham-operated(Sham) group and the four ovariectomized(OVX) subgroups. Rats subjected to Sham or OVX were administered with the vehicle(OVX, 1 m L water/100 g weight), 17β-estradiol(E2, 50 μg·kg-1·d-1), and lyophilized powder of ZGW at a low dose of 2.3(ZGW-L) and high dose of 4.6(ZGW-H) g·kg-1·d-1 for three months. The bone density and bone strength were assessed using dual-energy X-ray and three-point bending tests, respectively. Furthermore, enzyme-linked immunosorbent assay, Hematoxylin-eosin staining, and western blot analysis were used to determine the potential pharmacological mechanisms of action of ZGW in PMOP. RESULTS: A total of 117 active compounds of ZGW were screened from the TCMSP. Furthermore, 108 intersecting genes of drugs and diseases were identified. Using STRING software and the MCC algorithm, ten core genes, including C-X-C chemokine living 8(CXCL8), C-C chemokine receptor type 2(CCR2), alpha-2a active receptor(ADRA2A), melatonin receptor type 1B(MTNR1B), and amyloid-beta A4 protein(APP), were identified. The anti-osteoporosis regulation network of ZGW was constructed using the Cytoscape software. The animal experiments demonstrated that ZGW groups significantly reduced the serum levels of β-C-terminal telopeptide of type I collagen(β-CTX) and increased serum levels of bone-specific alkaline phosphatase(BALP)(P < 0.05, P < 0.01). The OVX group exhibited a significant decrease in bone mineral density and bone strength compared with the Sham group(P < 0.01). Moreover, treatment with ZGW resulted in increased trabecular thickness, improved arrangement of trabecular structure, and reduced empty bone lacunae. Furthermore, treatment with ZGW significantly increased the protein expression of CXCL8, ADRA2A, and CCR2(P < 0.05, P < 0.01), and significantly decreased the protein expression of Runx2(P < 0.01). Furthermore, the ZGW and E2 groups demonstrated significantly increased BMD(P < 0.05, P < 0.01), improved bone strength(P < 0.05, P < 0.01), reduced expression of CXCL8, ADRA2A, and CCR2, and increased runt-related transcription factor 2 levels in bone tissue(P < 0.05, P < 0.01) compared with the OVX group. However, there were no significant differences in MTNR1B and APP expression among the groups. CONCLUSION: ZGW shows synergistic mechanisms in PMOP through multiple components, targets, and pathways.展开更多
Objective:Zuogui Wan(ZGW)has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine.The objective of this study is to elucidate the phytochemical constituents of ZGW-treated r...Objective:Zuogui Wan(ZGW)has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine.The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum(ZGWRS)using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry(UPLC-ESI-Q-TOF-MS).Methods:ZGW was administered to rats,and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS.MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS.Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats.The fertilized eggs collected from the same experiment were randomly divided into four groups,including the normal,40 mmol/L glucose,40 mmol/L glucose 5%control rat serum,and 40 mmol/L glucose 5%ZGWRS groups.They were cultivated at 37°C and 5%CO2 in a saturated humidity CO2 incubator.The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development.Results:Thirteen constituents were identified in the ZGWRS,among which sweroside,loganin,morroniside,loganic acid,and 8-epiloganic acid were from Fructus Corni(Shan Zhu Yu).5-Hydroxymethyl-2-furfural-glucuronide,2-H-5-hydroxymethyl-2-furfural-glucuronide,3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid,andβ-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata(Shu Di Huang).Coumaric acid was from Fructus Lycii(Gou Qi Zi).Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae(Tu Si Zi).The embryonic development was significantly inhibited using 40 mmol/L glucose.Compared with the normal group,the blastocyst rate of the glucose group was decreased.The blastocyst rate of the 40 mmol/L glucose 5%ZGWRS group was significantly higher than that of the glucose group,indicating that ZGWRS negates the effect of glucose on mouse embryonic development.Conclusion:The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW.ZGWRS is rich in active constituents of iridoid glycosides.The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.展开更多
OBJECTIVE:To investigate the protective effects of Zuogui Wan(左归丸,ZGW)on bone loss induced by ovariectomy(OVX)and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model.METHODS:Fifty Sprague-...OBJECTIVE:To investigate the protective effects of Zuogui Wan(左归丸,ZGW)on bone loss induced by ovariectomy(OVX)and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model.METHODS:Fifty Sprague-Dawley female rats were randomly divided into sham-operated(sham)group and four OVX subgroups.Rats subjected to sham and OVX were treated with the vehicle(OVX,1 mL/100 g weight,n=10),17β-estradiol(E2,50μg·kg-1·d-1),and ZGW at the doses of 2.3(ZGW-L)and 4.6(ZGW-H)g/kg/day lyophilized powder daily for 3 months,respectively.The serum biochemical parameters of 17β-estrogen(17β-E2),tartrate-resistant acid phosphatase(TRACP-5 b)and bone alkaline phosphatase(BALP)were measured by enzyme-linked immunosorbent assay.Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones.Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur.The gene or protein expression of orexin-A,orexin receptor 1(OX1R),orexin receptor 2(OX2R),osteoprotegerin(OPG)and receptor activator of nuclear factor-κB ligand(RANKL)were assayed by either quantitative polymerase chain reaction or Western blot analysis.RESULTS:Compared with the OVX group,ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and 17β-E2(P<0.01).Meanwhile,ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number,and arranging the trabeculae structure properly.Compared with the OVX group,it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae,and OPG in the tibiae of ZGW groups(P<0.01,<0.05),while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae(P<0.01).CONCLUSION:ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.展开更多
基金the National Natural Science Foundation of China:Research on Mechanism of Zuogui Wan in Treating Postmenopausal Osteoporosis by Regulating Feed-forward Loop of Oxytocin/Oxytocin Receptor Based on Transcriptome so as to Explain the Theory of “All Marrows Dominated by Brain”(No. 82104730)Based on Protein Kinase Cθ/Nuclear Factor Kappa-B Signal Transduction Regulation,Shexiang Huangqi Compound Dropping Pills Regulate the Migration and Differentiation Mechanism of Mesenchymal Stem Cells Through the Blood-brain Barrier after Cerebral Ischemia (No. 81974564)+1 种基金The 72nd Batch of China Postdoctoral Science Foundation (2022M721065)Central Plains Talent Program-science and Technology Innovation Leading Talent Project (224200510027)。
文摘OBJECTIVE: To explore the multi-component synergistic mechanism of Zuogui Wan(左归丸, ZGW) in treating postmenopausal osteoporosis(PMOP). METHODS: The main components and target genes of ZGW were screened via the Traditional Chinese Medicine Systems Pharmacology(TCMSP). In addition, the target gene sets of PMOP were derived from the Gene Cards and Online Mendelian Inheritance in Man databases. The search tool for recurring instances of neighbouring genes(STRING) 11.0 software was used to analyze the interaction among intersecting genes. Cytoscape 3.6.1 software and the Matthews correlation coefficient(MCC) algorithm were used to screen the core genes. Fifty Sprague-Dawley female rats were randomly divided into the sham-operated(Sham) group and the four ovariectomized(OVX) subgroups. Rats subjected to Sham or OVX were administered with the vehicle(OVX, 1 m L water/100 g weight), 17β-estradiol(E2, 50 μg·kg-1·d-1), and lyophilized powder of ZGW at a low dose of 2.3(ZGW-L) and high dose of 4.6(ZGW-H) g·kg-1·d-1 for three months. The bone density and bone strength were assessed using dual-energy X-ray and three-point bending tests, respectively. Furthermore, enzyme-linked immunosorbent assay, Hematoxylin-eosin staining, and western blot analysis were used to determine the potential pharmacological mechanisms of action of ZGW in PMOP. RESULTS: A total of 117 active compounds of ZGW were screened from the TCMSP. Furthermore, 108 intersecting genes of drugs and diseases were identified. Using STRING software and the MCC algorithm, ten core genes, including C-X-C chemokine living 8(CXCL8), C-C chemokine receptor type 2(CCR2), alpha-2a active receptor(ADRA2A), melatonin receptor type 1B(MTNR1B), and amyloid-beta A4 protein(APP), were identified. The anti-osteoporosis regulation network of ZGW was constructed using the Cytoscape software. The animal experiments demonstrated that ZGW groups significantly reduced the serum levels of β-C-terminal telopeptide of type I collagen(β-CTX) and increased serum levels of bone-specific alkaline phosphatase(BALP)(P < 0.05, P < 0.01). The OVX group exhibited a significant decrease in bone mineral density and bone strength compared with the Sham group(P < 0.01). Moreover, treatment with ZGW resulted in increased trabecular thickness, improved arrangement of trabecular structure, and reduced empty bone lacunae. Furthermore, treatment with ZGW significantly increased the protein expression of CXCL8, ADRA2A, and CCR2(P < 0.05, P < 0.01), and significantly decreased the protein expression of Runx2(P < 0.01). Furthermore, the ZGW and E2 groups demonstrated significantly increased BMD(P < 0.05, P < 0.01), improved bone strength(P < 0.05, P < 0.01), reduced expression of CXCL8, ADRA2A, and CCR2, and increased runt-related transcription factor 2 levels in bone tissue(P < 0.05, P < 0.01) compared with the OVX group. However, there were no significant differences in MTNR1B and APP expression among the groups. CONCLUSION: ZGW shows synergistic mechanisms in PMOP through multiple components, targets, and pathways.
基金financially supported by the Shanxi Province Science Foundation for the National International Cooperation(201703D421031)Youth Fund of the National Natural Science Fund project(81903951)TCM discipline construction project of SXTCM(Direction 3)(1008Z3)。
文摘Objective:Zuogui Wan(ZGW)has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine.The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum(ZGWRS)using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry(UPLC-ESI-Q-TOF-MS).Methods:ZGW was administered to rats,and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS.MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS.Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats.The fertilized eggs collected from the same experiment were randomly divided into four groups,including the normal,40 mmol/L glucose,40 mmol/L glucose 5%control rat serum,and 40 mmol/L glucose 5%ZGWRS groups.They were cultivated at 37°C and 5%CO2 in a saturated humidity CO2 incubator.The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development.Results:Thirteen constituents were identified in the ZGWRS,among which sweroside,loganin,morroniside,loganic acid,and 8-epiloganic acid were from Fructus Corni(Shan Zhu Yu).5-Hydroxymethyl-2-furfural-glucuronide,2-H-5-hydroxymethyl-2-furfural-glucuronide,3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid,andβ-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata(Shu Di Huang).Coumaric acid was from Fructus Lycii(Gou Qi Zi).Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae(Tu Si Zi).The embryonic development was significantly inhibited using 40 mmol/L glucose.Compared with the normal group,the blastocyst rate of the glucose group was decreased.The blastocyst rate of the 40 mmol/L glucose 5%ZGWRS group was significantly higher than that of the glucose group,indicating that ZGWRS negates the effect of glucose on mouse embryonic development.Conclusion:The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW.ZGWRS is rich in active constituents of iridoid glycosides.The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.
基金Supported by Traditional Chinese and Western Medicine Clinical Medicine Brand Construction Project of Jiangsu Higher Education Institutions(PhaseⅡ)National Natural Science Foundation of China(No.81573874 and81873229)。
文摘OBJECTIVE:To investigate the protective effects of Zuogui Wan(左归丸,ZGW)on bone loss induced by ovariectomy(OVX)and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model.METHODS:Fifty Sprague-Dawley female rats were randomly divided into sham-operated(sham)group and four OVX subgroups.Rats subjected to sham and OVX were treated with the vehicle(OVX,1 mL/100 g weight,n=10),17β-estradiol(E2,50μg·kg-1·d-1),and ZGW at the doses of 2.3(ZGW-L)and 4.6(ZGW-H)g/kg/day lyophilized powder daily for 3 months,respectively.The serum biochemical parameters of 17β-estrogen(17β-E2),tartrate-resistant acid phosphatase(TRACP-5 b)and bone alkaline phosphatase(BALP)were measured by enzyme-linked immunosorbent assay.Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones.Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur.The gene or protein expression of orexin-A,orexin receptor 1(OX1R),orexin receptor 2(OX2R),osteoprotegerin(OPG)and receptor activator of nuclear factor-κB ligand(RANKL)were assayed by either quantitative polymerase chain reaction or Western blot analysis.RESULTS:Compared with the OVX group,ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and 17β-E2(P<0.01).Meanwhile,ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number,and arranging the trabeculae structure properly.Compared with the OVX group,it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae,and OPG in the tibiae of ZGW groups(P<0.01,<0.05),while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae(P<0.01).CONCLUSION:ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.
