Background:Bushen Zhuanggu Formula(BZF),derived from the classic Yougui Pills,has shown favorable clinical efficacy in treating advanced nontraumatic osteonecrosis of the femoral head(NONFH),particularly by promoting bone repair.However,its underlying mechanisms remain unclear.Objective:This study aimed to explore the mechanisms by which BZF promotes bone repair in advanced NONFH.Materials and methods:A total of 518 potential BZF targets were identified from the ETCM v2.0 database.Transcriptomic profiling of clinical cohorts revealed 485 differentially expressed genes in advanced NONFH patients compared to healthy controls.A drug target-disease gene interaction network was constructed to identify candidate BZF targets involved in NONFH pathogenesis.In vivo experiments were conducted to validate the effects of BZF in a rat model of advanced NONFH.Results:Network analysis identified key pathways associated with blood circulation obstruction,immune-inflammatory imbalance,and abnormal bone metabolism.Protein kinase C alpha(PKCA),Ras proto-oncogene(RAS),mitogen-activated protein kinase 3(ERK),ETS proto-oncogene 1(ETS1),and receptor activator of nuclear factor-κB ligand(RANKL)formed a signaling axis implicated in NONFH pathogenesis.BZF treatment alleviated joint inflammation,preserved trabecular bone morphology,reduced bone loss,and promoted bone repair.Mechanistically,BZF significantly downregulated the expression of PKCA,RAS,ERK,ETS1,and RANKL,improved blood circulation,and inhibited osteoclast activation while promoting osteoblast activation.Conclusion:BZF may promote bone repair in advanced NONFH by enhancing blood circulation and modulating the PKC-RAS-ERK-ETS1-RANKL signaling axis,thereby reversing dysregulated bone metabolism.

Background: Bushen Zhuanggu Formula (BZF), derived from the classic Yougui Pills, has shown favorable clinical efficacy in treating advanced nontraumatic osteonecrosis of the femoral head (NONFH), particularly by promoting bone repair. However, its underlying mechanisms remain unclear. Objective: This study aimed to explore the mechanisms by which BZF promotes bone repair in advanced NONFH. Materials and methods: A total of 518 potential BZF targets were identified from the ETCM v2.0 database. Transcriptomic profiling of clinical cohorts revealed 485 differentially expressed genes in advanced NONFH patients compared to healthy controls. A drug target-disease gene interaction network was constructed to identify candidate BZF targets involved in NONFH pathogenesis. In vivo experiments were conducted to validate the effects of BZF in a rat model of advanced NONFH. Results: Network analysis identified key pathways associated with blood circulation obstruction, immune-inflammatory imbalance, and abnormal bone metabolism. Protein kinase C alpha (PKCA), Ras proto-oncogene (RAS), mitogen-activated protein kinase 3(ERK), ETS proto-oncogene 1 (ETS1), and receptor activator of nuclear factor-κB ligand (RANKL) formed a signaling axis implicated in NONFH pathogenesis. BZF treatment alleviated joint inflammation, preserved trabecular bone morphology, reduced bone loss, and promoted bone repair. Mechanistically, BZF significantly downregulated the expression of PKCA, RAS, ERK, ETS1, and RANKL, improved blood circulation, and inhibited osteoclast activation while promoting osteoblast activation. Conclusion: BZF may promote bone repair in advanced NONFH by enhancing blood circulation and modulating the PKC-RAS-ERK-ETS1-RANKL signaling axis, thereby reversing dysregulated bone metabolism.

Background:Bushen Zhuanggu Formula(BZF),derived from the classic Yougui Pills,has shown favorable clinical efficacy in treating advanced nontraumatic osteonecrosis of the femoral head(NONFH),particularly by promoting bone repair.However,its underlying mechanisms remain unclear.Objective:This study aimed to explore the mechanisms by which BZF promotes bone repair in advanced NONFH.Materials and methods:A total of 518 potential BZF targets were identified from the ETCM v2.0 database.Transcriptomic profiling of clinical cohorts revealed 485 differentially expressed genes in advanced NONFH patients compared to healthy controls.A drug target-disease gene interaction network was constructed to identify candidate BZF targets involved in NONFH pathogenesis.In vivo experiments were conducted to validate the effects of BZF in a rat model of advanced NONFH.Results:Network analysis identified key pathways associated with blood circulation obstruction,immune-inflammatory imbalance,and abnormal bone metabolism.Protein kinase C alpha(PKCA),Ras proto-oncogene(RAS),mitogen-activated protein kinase 3(ERK),ETS proto-oncogene 1(ETS1),and receptor activator of nuclear factor-κB ligand(RANKL)formed a signaling axis implicated in NONFH pathogenesis.BZF treatment alleviated joint inflammation,preserved trabecular bone morphology,reduced bone loss,and promoted bone repair.Mechanistically,BZF significantly downregulated the expression of PKCA,RAS,ERK,ETS1,and RANKL,improved blood circulation,and inhibited osteoclast activation while promoting osteoblast activation.Conclusion:BZF may promote bone repair in advanced NONFH by enhancing blood circulation and modulating the PKC-RAS-ERK-ETS1-RANKL signaling axis,thereby reversing dysregulated bone metabolism.

Objective To establish the animal model of comorbidity of knee osteoarthritis(KOA)and hypertension with pattern of liver and kidney deficiency and evaluate its characteristics of comorbidity and pattern.Methods Wistar-Kyoto(WKY)rats and spontaneously hypertensive rats(SHR)were assigned to the WKY control group(control group),hypertension combined with KOA sham-operation group(sham-operation group),hypertension combined with KOA group(model group),hypertension combined with KOA and liver-kidney deficiency pattern group(LKD group).The animal model of KOA combined with hypertension was prepared by anterior cruciate ligament transection(ACLT)in spontaneously hypertensive rats.ACLT combined with intramuscular injection of hydrocortisone was performed to prepare an animal model of KOA with liver-kidney deficiency(LKD)pattern type,combined with hypertension.Then,the related indexes of LKD syndrome were detected in turn,including the contents of thyroid stimulating hormone(TSH),testosterone(T),corticosterone(CORT),adrenocorticotropic hormone(ACTH)in serum,and enzyme activities of alanine transaminase(ALT),aspartate transaminase(AST),and alkaline phosphatase(ALP)in serum,the mass ratio of liver,kidney,spleen,thymus to the brain,body weight,anal temperature,activity situation,and emotion.Systolic blood pressure,diastolic blood pressure,and other blood pressure-related indices were also detected.The levels of serum tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),plantar mechanical pain sensitivity threshold,weight difference score of both hind limbs,hind limb joint swelling,and quadruped gait parameters were also measured.Furthermore,hematoxylin-eosin,safranine-fast green,and Masson staining were performed to observe pathological changes,cartilage degeneration,and bone destruction of the knee joint,and the microstructure parameters of the tibia were detected by Micro-CT imaging.Results Compared to the model group,the contents of serum TSH,ACTH,T,CORT and the mass ratio of the kidney,spleen,and thymus to the brain in the LKD group decreased(P<0.05).Compared to the control group,the systolic blood pressure and diastolic blood pressure of the other three groups increased significantly(P<0.05).Compared to the sham-operation group,serum TNF-α and IL-1β levels increased,plantar mechanical pain threshold decreased,weight difference score of both hind limbs and joint swelling of the affected limb increased(P<0.05),and gait parameters(e.g.,gait length and standing time of the affected limbs)became abnormal in the model and LKD groups.Simultaneously,the cartilage surface defect of the rat knee joint was severe,the arrangement of the surface chondrocytes was altered,the cartilage layer became thinner,the muscle fibers increased,and the cartilage ossification was severe.Furthermore,the relative volume,thickness,and number of trabeculae of the knee joint decreased significantly(P<0.05).Conclusion The rat model established in this study is consistent with the clinical characteristics of integrated traditional Chinese and Western medicine in patients with comorbidities of hypertension and KOA with liver and kidney deficiency pattern.This rat model can characterize the typical symptoms of liver and kidney deficiency pattern.It has typical pathological changes in knee cartilage and subchondral bone tissues and can maintain a stable range of high systolic and diastolic blood pressure.It also explore the scientific connotation of simultaneous treatment of different diseases in traditional Chinese medicine,revealing the therapeutic mechanism and developing new drugs.

ObjectiveTo systematically and objectively characterize the pharmacological effects of Fufang Biejia Ruangan pills （FBRP） in the intervention of alcoholic liver disease （ALD） using acute and chronic ALD mouse models and to elucidate its molecular mechanisms. MethodFifty SPF-grade male BALB/c mice were randomly divided into the normal group， model group， and FBRP low-， medium-， and high-dose groups （9.6， 19.2， 38.4 mg·kg^-1）. Except for the normal group， the remaining groups were given 56° white wine by gavage to establish the acute ALD model， with samples collected after 4 weeks. Thirty SPF-grade male C57BL/6N mice were randomly divided into the normal group， model group， and FBRP medium-dose group （19.2 mg·kg^-1）. The chronic ALD mouse model was established using the Lieber-DeCarli method over a 10-week period. Inflammatory markers in liver tissues were assessed using hematoxylin-eosin （HE）， Sirius Red， oil red O staining， and enzyme-linked immunosorbent assay （ELISA）. Intoxication behaviors of each group were objectively evaluated through sobering-up time， net-catching， and pole-climbing tests. Further bioinformatics analyses based on clinical transcriptomic data were conducted to identify key targets and molecular mechanisms of FBRP in alleviating ALD through liver-brain dialogue， with experimental validation by ELISA， Western blot， and immunohistochemical staining. ResultCompared with the normal group, the levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） in liver tissues of mice in the acute and chronic ALD model groups were significantly increased （P<0.05）. Compared with the model group， the levels of AST and ALT in liver tissue of mice in FBRP groups were significantly decreased （P<0.05）. Compared with the normal group， the time of grasping the net and climbing the pole in the acute ALD model group was significantly decreased within 4 weeks （P<0.01）. Compared with the model group， the grasping and climbing time of FBRP high dose groups increased significantly within 4 weeks （P<0.05）. Compared with the normal group， the expression of high mobility group protein B1 （HMGB1） protein in liver tissue and prefrontal lobe tissue of mice in the chronic ALD model group was significantly increased （P<0.01）. Compared with the model group， the expression of HMGB1 protein in FBRP medium dose group was significantly decreased （P<0.05，P<0.01）. Compared with the normal group， the expression of brain-derived neurotrophic factor （BDNF） protein and the release of γ-aminobutyric acid （GABA） in the prefrontal cortex of the model group were significantly decreased （P<0.01）. Compared with the model group, the expression of BDNF protein and the release of GABA in the FBRP medium dose group were significantly increased （P<0.05）. ConclusionThis study revealed that FBRP improved key pathological changes in ALD by modulating liver-brain dialogue mediated by the HMGB1-BDNF axis. These findings provide experimental evidence for the clinical use of FBRP in treating ALD and offer new insights for the development of ALD therapeutic agents.

ObjectiveTo identify the pharmacodynamic material basis of Ruyi Zhenbaowan in relieving neuropathic pain by integrating the calculation of biological network proximity and pharmacokinetic characterization. MethodThe interaction network of "drug candidate target-related gene of disease" was constructed by Cytoscape 3.8.2， and the average shortest path value of each drug putative target acting on neuropathic pain-related genes in this network was calculated by Pesca 3.8.0 tool so as to evaluate the network proximity between them， and screen prescription candidate targets with strong intervention efficiency and their corresponding potential effect components. After that， plasma and cerebrospinal fluid samples were collected from rats after administration of Ruyi Zhenbaowan at set time points， and the contents of potential effect components in samples was quantified by ultra performance liquid chromatography-quadrupole-ion trap mass spectrometry（UPLC-Q-TRAP/MS）， and drug concentration-time curves were plotted， then the pharmacokinetic parameters were calculated by DAS 2.1.1. ResultBy evaluating the network proximity between candidate targets and neuropathic pain-related genes in the interaction network， a total of 40 putative targets of Ruyi Zhenbaowan with strong intervention effects on neuropathic pain-related genes， such as estrogen receptor 1（ESR1）， cyclic adenosine monophosphate（cAMP）-dependent protein kinase catalytic subunit alpha（PRKACA） and protein kinase B1 （Akt1）， and 10 corresponding potential effect components， such as glycyrrhizic acid and betulinic acid， were obtained. Pharmacokinetic characterization showed that among the 10 potential effect components， gallic acid， apigenin-7-O-glucuronide， glycyrrhizic acid and apigenin were well absorbed and metabolized in plasma and cerebrospinal fluid， with long onset time and good bioavailability. ConclusionFrom the perspective of efficacy-target-constituent-pharmacokinetic， this study analyzes the main effective materials of Ruyi Zhenbaowan， such as glycyrrhizic acid， gallic acid， apigenin-7-O-glucuronide and apigenin， which have a high exposure in plasma or cerebrospinal fluid and have a strong intervention effect on neuropathic pain. The related results provide reliable experimental evidences for clarifying the material basis and developing quality standards of Ruyi Zhenbaowan.

ObjectiveTo identify the chemical constituents of Ruyi Zhenbaowan in vitro and in vivo. MethodThe chemical constituents of Ruyi Zhenbaowan were identified based on UHPLC-Q Exactive Orbitrap HRMS. A total of 12 male SD rats were randomized into two groups： control （pure water） and Ruyi Zhenbaowan （1.8 g·kg^-1）. The rats were administrated with the suspension of Ruyi Zhenbaowan or pure water by gavage. After 1.5 h， the plasma and cerebrospinal fluid were collected. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C₁₈ column （2.1 mm × 150 mm， 1.7 μm） with a mixture of 0.1% formic acid aqueous solution （A） and acetonitrile （B） as the mobile phase. Gradient elution was carried out according to the procedure of 0~15 min，97%~80%A；15~30 min ，80%~60%A；30~40 min，60%~30%A；40~45 min，30%~5%A. The ion source was electrospray ionization， and scan range was m/z 100-1 500. The prototype components and the components in the plasma and cerebrospinal fluid were analyzed qualitatively by scanning in positive and negative ion modes and identified by comparison with the data in published literature and the information of standard substances. ResultA total of 126 chemical constituents were identified from the 80% methanol solution of Ruyi Zhenbaowan， and 14 and 7 prototype constituents were detected in the plasma and the cerebrospinal fluid， respectively. In addition， the fragmentation rules of apigenin， apigenin-7-O-glucuronide， galangin， liquiritin， piperine， glycyrrhizic acid， eugenol， gallic acid， and cholic acid were deduced. ConclusionThis study achieved rapid multicomponent characterization and identification of Ruyi Zhenbaowan in vitro and in vivo， providing theoretical support for exploring active substances and performing quality control.l.

ObjectiveTo elucidate the mechanism of Osteoking against fracture， femoral head necrosis， osteoarthritis， and lumbar disc herniation by integrating heterogeneous information network mining and experimental validation. MethodOn the basis of the disease-related database and transcriptome expression profiling dataset， as well as the ETCM database， the gene sets related to four target diseases and the candidate target spectrum of Osteoking were obtained through the integration and analysis of bioinformatics data， and a "disease-syndrome-formula-target-pathway-effect" heterogeneous information network was constructed. In addition， by functional enrichment analysis， the core targets of Osteoking in interfering with the imbalance network of four kinds of bone injury diseases， the biological pathways involved， and the corresponding clinical symptoms were screened， and they were verified in animal experiments. ResultHeterogeneous information network mining indicates that Osteoking may commonly reverse the imbalance networks of fracture， femoral head necrosis， osteoarthritis， and lumbar disc herniation via regulating cell function and activity， inhibiting inflammatory response， reducing bone destruction， and improving the immune function of the body by modulating relevant core candidate targets such as RAC-alpha serine/threonine-protein kinase （Akt1）， catenin beta-1 （CTNNB1）， epidermal growth factor receptor （EGFR）， heat shock protein 90-alpha （HSP90AA1）， and phosphatidylinositol 3-kinase catalytic subunit alpha isoform （PI3KCA）， as well as related biological pathways such as phosphatidylinositide 3-kinases/protein kinase B （PI3K/Akt）， janus kinase/signal transducer and activator of transcription （JAK/STAT）， tumor necrosis factor （TNF）， nuclear factor kappa-B （NF-κB）， and Toll-like receptors. In particular， Osteoking may improve the blood supply of the fracture end by regulating blood circulation at the target site of the disease， and it may maintain the balance of bone metabolism by regulating hormone-related pathways to promote fracture healing. In addition， Osteoking may relieve lipid metabolism disorders by targeting and regulating lipid-related pathways， accelerate bone formation and bone repair， and delay the progression of femoral head necrosis. Osteoking may relieve the symptoms of pain by acting on neurological pathways to reduce local nociceptive stimulation in patients with osteoarthritis and lumbar disc herniation. Further experimental validation demonstrates that the PI3K/Akt signaling pathway is the most significantly enriched pathway for the key network targets of Osteoking for the four diseases. The candidate target of Osteoking may have the strongest association with the network of fracture-related genes. Therefore， this study chooses fracture as the target disease to verify the efficacy of Osteoking. The results show that Osteoking can accelerate bone formation and promote fracture healing by inhibiting the activation of the PI3K/Akt signaling axis. ConclusionThe study shows that the main mechanism of "treating different diseases with an identical treatment" of four bone injury diseases with Osteoking involves cell function regulation and immune inflammation-related signaling pathways. Further experimental validation identifies that the PI3K/Akt signaling axis may be one of the key pathways of Osteoking to promote bone regeneration， bone reconstruction， and bone metabolism homeostasis.