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.

A strategy combining a tailored database and high-throughput activity screening that discover bioactive metabolites derived from Magnoliae Officinalis Cortex (MOC) was developed and implemented to rapidly profile and discover bioactive metabolites in vivo derived from traditional Chinese medicine (TCM). The strategy possessed four characteristics: 1) The tailored database consisted of metabolites derived from big data-originated reference compound, metabolites predicted in silico, and MOC chemical profile-based pseudomolecular ions. 2) When profiling MOC-derived metabolites in vivo, attentions were paid not only to prototypes of MOC compounds and metabolites directly derived from MOC compounds, as reported by most papers, but also to isomerized metabolites and the degradation products of MOC compounds as well as their derived metabolites. 3) Metabolite traceability was performed, especially to distinguish isomeric prototypes-derived metabolites, prototypes of MOC compounds as well as phase I metabolites derived from other MOC compounds. 4) Molecular docking was utilized for high-throughput activity screening and molecular dynamic simulation as well as zebrafish model were used for verification. Using this strategy, 134 metabolites were swiftly characterized after the oral administration of MOC to rats, and several metabolites were reported for the first time. Furthermore, 17 potential active metabolites were discovered by targeting the motilin, dopamine D2, and the serotonin type 4 (5-HT₄) receptors, and part bioactivities were verified using molecular dynamic simulation and a zebrafish constipation model. This study extends the application of mass spectrometry (MS) to rapidly profile TCM-derived metabolites in vivo, which will help pharmacologists rapidly discover potent metabolites from a complex matrix.

A strategy combining a tailored database and high-throughput activity screening that discover bioactive metabolites derived from Magnoliae Officinalis Cortex(MOC)was developed and implemented to rapidly profile and discover bioactive metabolites in vivo derived from traditional Chinese medicine(TCM).The strategy possessed four characteristics:1)The tailored database consisted of metabolites derived from big data-originated reference compound,metabolites predicted in silico,and MOC chemical profile-based pseudomolecular ions.2)When profiling MOC-derived metabolites in vivo,attentions were paid not only to prototypes of MOC compounds and metabolites directly derived from MOC compounds,as reported by most papers,but also to isomerized metabolites and the degradation products of MOC compounds as well as their derived metabolites.3)Metabolite traceability was performed,especially to distinguish isomeric prototypes-derived metabolites,prototypes of MOC compounds as well as phase Ⅰ metabolites derived from other MOC compounds.4)Molecular docking was utilized for high-throughput activity screening and molecular dynamic simulation as well as zebrafish model were used for verification.Using this strategy,134 metabolites were swiftly characterized after the oral administration of MOC to rats,and several metabolites were reported for the first time.Furthermore,17 potential active metabolites were discovered by targeting the motilin,dopamine D2,and the serotonin type 4(5-HT4)receptors,and part bioactivities were verified using molecular dynamic simulation and a zebrafish constipation model.This study extends the application of mass spectrometry(MS)to rapidly profile TCM-derived metabolites in vivo,which will help pharmacologists rapidly discover potent metabolites from a complex matrix.

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.

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.

Objective:To analyze the clinical characteristics of gastrointestinal symptoms and liver function injury in patients with coronavirus disease 2019 (COVID-19).Methods:From January 23, 2020 to February 29, 2020, the medical records of 251 patients with COVID-19 admitted to the West Campus of the Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, were collected. The proportion of the patients with gastrointestinal symptoms including anorexia, nausea and vomiting, diarrhea and abdominal pain were analyzed respectively. The patients were divided into common type (76 cases), severe type (65 cases) and critical type (110 cases). The incidence of liver function injury and the changes of liver function parameters such as total bilirubin (TBil), direct bilirubin (DBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), albumin and globulin of the patients with different clinical types and with or without gastrointestinal symptoms were analyzed. Mann-Whitney U test, Chi square test and Fisher′s exact test were used for statistical analysis. Results:The main gastrointestinal symptoms of patients with COVID-19 were anorexia (33.9%, 85/251), diarrhea (12.0%, 30/251), nausea and vomiting (7.6%, 19/251) and abdominal pain (1.2%, 3/251). 143 patients (57.0%) had liver function injury, the rate of liver function injury in critical type patients was 75.5% (83/110), which was higher than that of common type patients (40.8%, 31/76) and severe type patients (44.6%, 29/65), and the differences were statistically significant ( χ2=22.765 and 16.865, both P<0.01). There was no significant difference in the proportion of patients with liver function injury between common type and severe type patients ( P>0.05). There was no statistically significant difference in the proportion of liver function injury between patients with gastrointestinal symptoms and those without gastrointestinal symptoms (57.8%(67/116) vs. 56.3%(76/135), P>0.05). The median values of TBil, DBil, ALT, AST, ALP, GGT, LDH and globulin level of critical type patients were 13.5 μmol/L, 4.9 μmol/L, 44.5 U/L, 50.0 U/L, 64.0 U/L, 41.0 U/L, 527.0 U/L and 33.6 g/L respectively. The proportions of critical type patients with TBil level >34.2 μmol/L, DBil level>13.6 μmol/L, ALT level>80 U/L and AST level>80 U/L were 7.3% (8/110), 7.3% (8/110), 17.3% (19/110) and 17.3% (19/110), respectively. These results were all higher than those of common type patients (9.5 μmol/L, 2.9 μmol/L, 28.5 U/L, 28.5 U/L, 54.0 U/L, 25.5 U/L, 225.5 U/L, 30.1 g/L, 0, 0, 6.6% (5/76) and 2.6% (2/76) ) and severe type patients (10.4 μmol/L, 3.4 μmol/L, 30.0 U/L, 31.0 U/L, 49.0 U/L, 25.0 U/L, 284.0 U/L, 30.7 g/L, 0, 0, 6.2% (4/65) and 1.5% (1/65)), and the differences were statistically significant ( Z=-4.264, -5.507, -4.000, -6.558, -3.112, -4.333, -4.858, -3.873, Fisher′s exact test, Fisher′s exact test, χ2=4.574, 9.620; Z=-3.060, -3.850, -3.923, -5.005, -9.495, -7.651, -3.853, -2.725, Fisher′s exact test, Fisher′s exact test, χ2=4.425, 10.169; all P<0.01). The median values of pre-albumin level, albumin level and the albumin to globulin ratio of critical type patients were 85.3 g/L, 28.2 g/L and 0.8, which were all lower than those of common type patients (157.3 g/L, 32.3 g/L and 1.1, respectively) and severe type patients (133.6 g/L, 31.6 g/L and 1.1, respectively), and the differences were statistically significant ( Z=-6.631, -3.647, -4.924, -4.503, -5.283 and -3.903, all P<0.01). The median albumin level of patients with diarrhea was lower than that of patients without diarrhea (28.2 g/L vs. 30.5 g/L), the proportion of diarrhea patients whose TBil level >20.0 to 34.2 μmol/L was higher than that of patients without diarrhea (70.0%, 21/30 vs. 10.9%, 24/221), and the differences were statistically significant ( Z=-2.182, χ2 =62.788; both P<0.05). Conclusions:Anorexia is the most common digestive symptom in COVID-19 patients, and the incidences of abdominal pain is low. The incidence of liver function injury of critical type patients is high. There is no significant correlation between gastrointestinal symptoms and liver function injury, and patients with diarrhea have lower albumin levels.

Objective To establish a laparoscopic classification of extrahepatic biliary dilatations (EHBD) that can guide minimally invasive treatment.Methods According to inclusion criteria,124 patients with EHBD who were admitted and treated from July 2001 to July 2017 in the First Hospital Affiliated to Army Military Medical University were included in this study.A new laparoscopic classification of EHBD was proposed based on the preoperative imaging data and laparoscopic findings of the position and extent of EHBD.The minimally invasive diagnosis and treatment strategies were made based on the new classification.Results According to the preoperative imaging data and intraoperative laparoscopic findings,124 patients with EHBD were divided into the following groups:type A (upper segment,34 cases),type B (middle segment,27 cases),type C (lower segment,20 cases),and type D (entire bile duct,43 cases).The clinical symptoms (abdominal pain,jaundice and mass) and reoperation rates were not significantly different among the 4 groups(both P＞0.05).The incidences of comorbidities (calculus or inflammation) were significantly different (P＜0.05).The operative time(type A:237.6±66.7 min,type B:259.2±60.0 min,type C:286.1 ± 74.7 min,type D:347.5±94.4 min) and blood loss (type A:192.6±102.2 ml,type B:201.5±120.2 ml,type C:297.5±162.1 ml,type D:305.8±237.3 ml) were significantly different among the groups (P＜ 0.05).The short-term complication rates after surgery (5.9％ ～ 20.0％) were significantly different (P＜ 0.05),while the long-term complication rates after surgery (7.4％ ～ 10.0％) were not significantly different.The conversion rates to open surgery were significantly higher in patients with type C and D lesions than in those with type A and B lesions (P＜0.05).Conclusion This laparoscopic classification predicted the difficulty of laparoscopic surgery for EHBD and had a guiding significance in the minimally invasive treatment for this disease entity.