ObjectiveTo systematically identify the chemical constituents of Xuefu Zhuyutang（XFZY） and quantitatively determine its main components， aiming to elucidate its pharmacodynamic material basis and provide a scientific foundation for improving its quality control standards. MethodsUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was employed for qualitative analysis of XFZY， and the identification of compounds was accomplished by comparing their retention times， secondary MS fragment ion information， 52 reference standards and relevant databases， followed by attribution of their herbal sources. A total of 22 representative compounds were screened out， and UPLC-quadrupole-linear ion trap mass spectrometry（UPLC-Q-TRAP-MS/MS） was applied for quantitative analysis of the compounds in the formula. ResultsA total of 77 compounds were identified in XFZY， including 31 flavonoids mainly derived from Aurantii Fructus， Glycyrrhizae Radix et Rhizoma， Persicae Semen， Carthami Flos， Bupleuri Radix， Paeoniae Radix Rubra and Achyranthis Bidentatae Radix， 24 terpenoids mainly derived from Platycodonis Radix， Glycyrrhizae Radix et Rhizoma， Paeoniae Radix Rubra and Rehmanniae Radix， 9 phenylpropanoids and their derivatives mainly derived from Chuanxiong Rhizoma， Angelicae Sinensis Radix and Rehmanniae Radix， 4 phenolic acids mainly derived from Chuanxiong Rhizoma， Angelicae Sinensis Radix and Paeoniae Radix Rubra， 3 saccharides mainly derived from Rehmanniae Radix and Achyranthis Bidentatae Radix， and 6 other compounds mainly derived from Persicae Semen， Rehmanniae Radix and Angelicae Sinensis Radix. The results of quantitative analysis showed that the contents of protocatechuic acid， hydroxypaeoniflorin， amygdalin， vanillic acid， paeoniflorin， liquiritin apioside， liquiritin， isoquercitrin， naringin， cosmosiin， hesperidin， neohesperidin， isoliquiritin， liquiritigenin， naringenin， benzoylpaeoniflorin， hesperetin， isoliquiritigenin， formononetin， glycyrrhizic acid， nobiletin and ligustilide in XFZY were determined to be 0.12， 1.57， 54.53， 0.29， 36.17， 4.29， 4.84， 0.09， 46.67， 0.04， 3.44， 31.95， 0.82， 0.10， 0.11， 0.43， 0.07， 0.03， 0.01， 8.24， 0.13， 1.81 mg·g^-1. ConclusionThe qualitative method established in this study enables rapid and sensitive analysis of the chemical constituents in XFZY. Among the identified compounds， 52 are confirmed by reference standards， ensuring the accuracy of identification. The quantitative analysis of 22 key components provides a reliable experimental basis for the pharmacodynamic material basis research and quality control standard improvement of XFZY.

OBJECTIVE To establish a method for determining the contents of clopidogrel （CLP）， clopidogrel carboxylate （CLP-C）， clopidogrel acyl-β-D-glucuronide （CLP-G） and contents of clopidogrel active metabolite （CAM） in rat plasma， and to investigate their in vivo pharmacokinetic characteristics. METHODS The Shisedo CAPCELL ADME column was used with a mobile phase consisting of water and acetonitrile （both containing 0.1% formic acid） in a gradient elution. The flow rate was 0.4 mL/min， and the column temperature was maintained at 20 ℃. The injection volume was 2 μL. The analysis was performed in positive ion mode using electrospray ionization with multiple reaction monitoring. The ion pairs for quantitative analysis were m/z 322.1→211.9 （for CLP）， m/z 308.1→197.9 （for CLP-C）， m/z 322.1→154.8 （for CLP-G）， m/z 504.1→154.9 [for racemic CAM derivative （CAMD）]. Six rats were administered a single intragastric dose of CLP （10 mg/kg）. Blood samples were collected before medication and at 0.08， 0.33， 0.66， 1， 2， 4， 6， 10， 23 and 35 hours after medication. The established method was used to detect the serum contents of various components in rats. Pharmacokinetic parameters were then calculated using WinNonlin 6.1 software. RESULTS The linear ranges for CLP， CLP-C and CAMD were 0.08-20.00， 205.00-8 000.00， and 0.04-25.00 ng/mL， respectively （r≥0.990）. The relative standard deviations for both intra-day and inter-day precision tests were all less than 15%， and the relative errors for accuracy ranged from －11.68% to 14.40%. The coefficients of variation for the matrix factors were all less than 15%， meeting the requirements for bioanalytical method validation. The results of the pharmacokinetic study revealed that， following a single intagastric administration of CLP in rats， the exposure to the parent CLP in plasma was extremely low. Both the area under the drug concentration-time curve （AUC0-35 h） and the peak concentration of the parent CLP were lower than those of its metabolites. The AUC0-35 h of the active metabolite CAM was approximately 43 times that of CLP， though it had a shorter half-life （2.53 h）. The inactive metabolite CLP-C exhibited the highest exposure level， but it reached its peak concentration the latest and was eliminated slowly. The AUC0-35 h of CLP-G was about four times that of CAM， and its half-life was similar to that of CLP-C. CONCLUSIONS This study successfully established an liquid chromatography-tandem mass spectrometry method for the determination of CLP and its three metabolites， and revealed their pharmacokinetic characteristics in rats. Specifically， the parent drug CLP was rapidly eliminated， while the inactive metabolites CLP-C and CLP-G exhibited long half-lives， and active metabolite CAM displayed a transient exposure pattern.

This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

Objective To investigate the clinical efficacy of Qi Shi Shen Shu Capsule combined with dapagliflozin in treating type 2 diabetic nephropathy.Methods 120 patients with type 2 diabetic nephropathy(qi-yin liangxu syndrome)admitted to the endocrinology department in the inpatient and outpatient depart-ments of endocrinology from May 2022 to December 2023 were randomly divided into control and observation groups(60 cases each).The control group received dapagliflozin plus placebo capsules,while the observation group received dapagliflozin plus Qi Shi Shen Shu Capsule for 6 months.The therapeutic effects were observed between the two groups.Parameters including serum creatinine(Scr),urinary albumin-to-creatinine ratio(uACR),24-hour urinary protein quantification(24hUP),glycated hemoglobin(HbA1c),and tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and high sensitivity-C reactive protein(hs-CRP)were measured be-fore and after treatment.Results The total effective rate was higher(P＜0.05)in the observation group(93.33％)than in the control group(78.33％).After treatment,both groups showed decreased levels of uACR,Scr,24hUP,HbA1c,TNF-α,IL-6,and hs-CRP compared with baseline,with lower levels in the obser-vation group(P＜0.05).Conclusion Qi Shi Shen Shu Capsule combined with dapagliflozin can effectively re-duce uACR,Scr levels and serum inflammatory cytokines levels in patients with type 2 diabetic nephropathy.

Objective To analyze the influence of cyclic RNA homologous domain interacting protein kinase 3(circ_HIPK3)on function and morphology of myloid β-protein(Aβ)induced hippocampal neurons by targeting miR-381-3p/zinc finger protein 217(ZNF217)axis.Methods Hippocampal neurons of neonatal rats were prepared and divided into the control group,the Aβ group,the si NC1 group,the si HIPK3 group,the si HIPK3+inhibitor NC group,the si HIPK3+miR-381-3p inhibitor group,the si HIPK3+miR-381-3p inhibitor+si NC2 group and the si HIPK3+miR-381-3p inhibitor+si ZNF217 group.Except the control group,all the other groups were modeled by 40 μmol/L Aβ1～42.qRT-PCR was used to determine the circ of hippocampal neurons circ_HIPK3,miR-381-3p and ZNF217 mRNA levels.Cell morphology was observed by transmission electron microscope,and the survival rate of hippocampal neurons was measured by CCK-8 method.Hochesst 33342 method was used to measure apoptosis of hippocampal neurons.The intracellular Ca2+ fluorescence intensity of hippocampal neurons was detected by flow cytometry.The expression levels of P-Tau,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Caspase-3 and ZNF217 proteins in hippocampal neurons were measured by Western blot assay.Double luciferase reporter genes were used to analyze the targeting relationship between miR-381-3p and circ_HIPK3,ZNF217.Results In the control group,the structure of hippocampal neurons was normal,the morphology of nucleus was normal,and there were no pathological changes in mitochondria and endoplasmic reticulum.In the Aβ group,hippocampal neurons showed degenerative changes,abnormal nuclear morphology,membrane invagination,a large number of mitochondria swelling and a large number of lipid droplets vacuoles in cytoplasm.Compared with the Aβ group,the hippocampal neuronal structure was partially restored in the si HIPK3 group.Compared with the si HIPK3 group,the hippocampal neuronal structure was severely damaged in the si HIPK3+miR-381-3p inhibitor group.Compared with the si HIPK3+miR-381-3p inhibitor group,the damage of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group was reduced.Compared with the control group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax,Caspase-3 protein expression of hippocampal neurons were increased in the Aβ group,and the miR-381-3p level,survival rate and Bcl-2 protein expression decreased(P＜0.05).Compared with the Aβ group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons were decreased in the si HIPK3 group,and miR-381-3p level,survival rate and Bcl-2 protein expression increased(P＜0.05).Compared with the si HIPK3 group,the circ_HIPK3,ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor group were increased,and the miR-381-3p level,survival rate and Bcl-2 protein expression decreased(P＜0.05).Compared with the si HIPK3+miR-381-3p inhibitor group,the ZNF217 mRNA and ZNF217 protein levels,apoptosis rate,Ca2+ fluorescence intensity,P-Tau,Bax and Caspase-3 protein expression of hippocampal neurons in the si HIPK3+miR-381-3p inhibitor+si ZNF217 group were decreased,and the survival rate and Bcl-2 protein expression increased(P＜0.05).miR-381-3p targeted and combined with HIPK3 and ZNF217.Conclusion circ_HIPK3 silencing may ameliorate Aβ-induced damage of hippocampal neuronal structure and function by regulating miR-381-3p/ZNF217 axis.

Objective To explore the relationship between preoperative coronary angiography and postoperative acute kidney injury (AKI) in cardiac surgery. Methods The clinical data of patients who underwent coronary angiography within 30 days before cardiac surgery in the First Affiliated Hospital of Xi’an Jiaotong University from January 2015 to April 2019 were retrospectively analyzed. Univariate analysis and multivariate logistic regression analyses were used to explore the relationship between the interval from preoperative coronary angiography to cardiac surgery and postoperative AKI. Results Finally 1 112 patients were collected, including 700 males and 412 females, with a median age of 61 (55, 66) years. The incidence of postoperative AKI was 40.8% (454/1 112), of which grade 2-3 AKI accounted for 11.9%. Multivariate analysis showed that age (OR=1.049, 95%CI 1.022-1.077, P<0.001), body mass index (OR=1.065, 95%CI 1.010-1.123, P=0.020) and time interval between preoperative coronary angiography and cardiac surgery within 24 hours (OR=1.625, 95%CI 1.116-2.364, P=0.011) were independent predictors of postoperative AKI. Patients who underwent coronary angiography within 24 hours before surgery had a 10.6% higher incidence of postoperative AKI compared to those who underwent angiography ≥24 hours before surgery (P=0.004). Patients who underwent valve surgery with or without coronary artery bypass grafting (CABG) had a higher risk of AKI than those who only underwent CABG. The in-hospital stay of patients who developed AKI was 2 days longer than those without AKI. However, undergoing coronary angiography within 24 hours before cardiac surgery did not prolong the length of ICU stay or hospital stay, nor did it increase the risk of death or renal failure after the operation. Conclusion Undergoing coronary angiography within 24 hours before cardiac surgery increases the risk of postoperative AKI.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.

Objective:To evaluate the effect of preemptive analgesia with ibuprofen on postoperative pain following single posterior tooth implantation, aiming to provide a clinical reference for its application.Methods:A multicenter, randomized, double-blind, placebo-controlled parallel-group trial was conducted. A total of 82 participants were included in the trial, meeting the eligibility criteria from April 2022 to April 2024 at the Capital Medical University School of Stomatology (40 cases), Beijing TianTan Hospital, Capital Medical University (22 cases), Beijing Chao-Yang Hospital, Capital Medical University (20 cases). Participants were randomly assigned in a 1∶1 ratio to either the ibuprofen group or the control group, with each group comprising 41 individuals. Participants in the ibuprofen group received 300 mg of sustained-release ibuprofen capsules orally 15 min before surgery, while the control group received a placebo. Both groups received the same postoperative analgesic regimen for 3 days. Pain scores were assessed using the numerical rating scale at 30 min, 4 h, 6 h, 8 h, 24 h, 48 h, and 72 h postoperatively, and the additional use of analgesic medication was recorded from days 4 to 6 postoperatively.Results:A total of 82 participants were initially enrolled in the study, with 7 dropouts (4 from the control group and 3 from the ibuprofen group), resulting in 75 participants (37 in the control group and 38 in the ibuprofen group) completing the trial. There were no reports of adverse events such as nausea or vomiting among the participants. The ibuprofen group exhibited significantly lower pain scores at 4 h, 6 h and 8 h [1.0 (0.0, 2.0), 1.0 (0.0, 2.0), 1.5 (0.0, 3.0) ] postoperatively compared to the control group 4 h, 6 h and 8 h [2.0 (1.0, 3.0), 3.0 (1.5, 4.0), 2.0 (1.0, 4.0)] ( Z=-1.99, P=0.047; Z=-3.01, P=0.003; Z=-2.10, P=0.036). The proportions of patients requiring additional analgesic medication between days 4 and 6 post-surgery were 18.4% (7/38) in the ibuprofen group and 27.0% (10/37) in the control group, with no significant difference (χ 2=0.79, P=0.373). The median additional medication usage postoperatively was [0.0 (0.0, 0.0) pills] in the ibuprofen group and [0.0 (0.0, 1.0) pills] in the control group, with no significant difference ( Z=-0.78, P=0.439). Conclusions:Preemptive analgesia with ibuprofen effectively reduces postoperative pain following tooth implantation, representing a safe and effective perioperative pain management strategy.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.