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.

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 evaluate the risk of right heart failure after heart transplantation by establishing nomogram based on preoperative pulmonary artery pressure.Methods A total of 184 patients undergoing heart transplantation were retrospectively collected and divided into the training group(126 cases)and the verification group(58 cases).Patients in the training set were divided into the right heart failure group(60 cases)and the non-right heart failure group(66 cases)according to whether right heart failure occurred after operation.The differences of clinical data between the two groups were compared,and the influencing factors of right heart failure occurred after operation in the training set were screened by Lasso-Logistic regression.According to the screened influencing factors,nomograms were drawn,and the predictive efficiency of the model was evaluated by using the receiver's operating characteristic(ROC)curve,calibration curve,receiver's operating characteristic(ROC)curve and clinical decision curve.Further vertification of the clinical application effect of centralized evaluation model was conducted.Results The Lasso-Logistic regression analysis identified the following independent risk factors for right heart failure after heart transplantation:elevated total bilirubin(OR=2.649,95％CI:1.339-5.239),increased mean pulmonary artery pressure(OR=3.082,95％CI:1.608-5.910),elevated pulmonary artery resistance(OR=3.171,95％CI:1.710-5.879),and widened right ventricular outflow tract diameter(OR=2.681,95％CI:1.361-5.281),all of which demonstrated statistical significance(P＜0.05).The nomogram model was constructed accordingly.The AUC of the nomogram model was 0.846(95％CI:0.813-0.947).The calibration curve demonstrated good fit via the goodness-of-fit test(Hosmer-Lemeshow x2=0.862,P=0.361).Clinical decision curve analysis revealed that the net benefit rate remained＞0 when the high-risk threshold probability ranged from 1％to 95％,indicating favorable clinical utility of this nomogram model.Based on the model predictions,among 58 heart transplant patients in the validation cohort,34 were classified as high-risk for right heart failure and 24 as low-risk.Actual diagnosis results showed 29 cases with right heart failure and 29 without.The Kappa coefficient reached 0.483(95％CI:0.261-0.705),demonstrating high consistency between model predictions and actual clinical outcomes.Conclusion Preoperative pulmonary systolic pressure increase is an independent risk factor for right heart failure after heart transplantation.A nomogram prediction model for right heart failure after heart transplantation is established by combining other clinical risk factors,and it has good prediction efficiency.

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 evaluate the risk of right heart failure after heart transplantation by establishing nomogram based on preoperative pulmonary artery pressure.Methods A total of 184 patients undergoing heart transplantation were retrospectively collected and divided into the training group(126 cases)and the verification group(58 cases).Patients in the training set were divided into the right heart failure group(60 cases)and the non-right heart failure group(66 cases)according to whether right heart failure occurred after operation.The differences of clinical data between the two groups were compared,and the influencing factors of right heart failure occurred after operation in the training set were screened by Lasso-Logistic regression.According to the screened influencing factors,nomograms were drawn,and the predictive efficiency of the model was evaluated by using the receiver's operating characteristic(ROC)curve,calibration curve,receiver's operating characteristic(ROC)curve and clinical decision curve.Further vertification of the clinical application effect of centralized evaluation model was conducted.Results The Lasso-Logistic regression analysis identified the following independent risk factors for right heart failure after heart transplantation:elevated total bilirubin(OR=2.649,95％CI:1.339-5.239),increased mean pulmonary artery pressure(OR=3.082,95％CI:1.608-5.910),elevated pulmonary artery resistance(OR=3.171,95％CI:1.710-5.879),and widened right ventricular outflow tract diameter(OR=2.681,95％CI:1.361-5.281),all of which demonstrated statistical significance(P＜0.05).The nomogram model was constructed accordingly.The AUC of the nomogram model was 0.846(95％CI:0.813-0.947).The calibration curve demonstrated good fit via the goodness-of-fit test(Hosmer-Lemeshow x2=0.862,P=0.361).Clinical decision curve analysis revealed that the net benefit rate remained＞0 when the high-risk threshold probability ranged from 1％to 95％,indicating favorable clinical utility of this nomogram model.Based on the model predictions,among 58 heart transplant patients in the validation cohort,34 were classified as high-risk for right heart failure and 24 as low-risk.Actual diagnosis results showed 29 cases with right heart failure and 29 without.The Kappa coefficient reached 0.483(95％CI:0.261-0.705),demonstrating high consistency between model predictions and actual clinical outcomes.Conclusion Preoperative pulmonary systolic pressure increase is an independent risk factor for right heart failure after heart transplantation.A nomogram prediction model for right heart failure after heart transplantation is established by combining other clinical risk factors,and it has good prediction efficiency.

Objective:To analyze the effect of emodin on apoptosis and oxidative damage of PC12 cells induced by hypoxia/re-oxygenation and its corresponding mechanism.Methods:PC12 cells were cultured in vitro and their neural differentiation was induced.Cells were divided into control group,model group,low concentration emodin group(30 mmol/L),high concentration emodin group(90 mmol/L)and high concentration emodin+GW441756(90 mmol/L+6.3 μmol/L)group,excepted for control group,other groups were treated with hypoxia and reoxygenation,and viability of PC12 cells was determined by CCK-8 method;levels of LDH release,SOD,MDA,IL-1β and TNF-α were measured by kits;PC12 cell apoptosis was detected by flow cytometry,expressions of nerve growth factor(NGF)/tropomyosin receptor kinase A(TrkA)pathway proteins in PC12 cells were determined by Western blot.Results:Compared with control group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in model group were decreased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were increased(P<0.05);compared with model group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in low concentration emodin group and high concentration emodin group were increased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were decreased(P<0.05);compared with high concen-tration emodin group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in high concentration emodin+GW441756 group were decreased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were increased(P<0.05).Conclusion:Emodin can inhibit oxidative stress and inflammatory damage of PC12 cells induced by hypoxia/reoxygenation,and inhibit apoptosis by activating NGF/TrkA pathway.

Objective:To analyze the effect of emodin on apoptosis and oxidative damage of PC12 cells induced by hypoxia/re-oxygenation and its corresponding mechanism.Methods:PC12 cells were cultured in vitro and their neural differentiation was induced.Cells were divided into control group,model group,low concentration emodin group(30 mmol/L),high concentration emodin group(90 mmol/L)and high concentration emodin+GW441756(90 mmol/L+6.3 μmol/L)group,excepted for control group,other groups were treated with hypoxia and reoxygenation,and viability of PC12 cells was determined by CCK-8 method;levels of LDH release,SOD,MDA,IL-1β and TNF-α were measured by kits;PC12 cell apoptosis was detected by flow cytometry,expressions of nerve growth factor(NGF)/tropomyosin receptor kinase A(TrkA)pathway proteins in PC12 cells were determined by Western blot.Results:Compared with control group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in model group were decreased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were increased(P<0.05);compared with model group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in low concentration emodin group and high concentration emodin group were increased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were decreased(P<0.05);compared with high concen-tration emodin group,PC12 cell activity,SOD level,NGF,and p-TrkA/TrkA in high concentration emodin+GW441756 group were decreased(P<0.05),while LDH release,apoptosis rate,MDA,IL-1β and TNF-α levels were increased(P<0.05).Conclusion:Emodin can inhibit oxidative stress and inflammatory damage of PC12 cells induced by hypoxia/reoxygenation,and inhibit apoptosis by activating NGF/TrkA pathway.

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.

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.