Objective To investigate the effect of fraxetin(FXT)on the malignant biological behavior of ovarian cancer cells by regulating Toll-like receptor 4(TLR4)/signal transducer and activator of transcription 3(STAT3)pathway.Methods Ovarian cancer cell lines SKOV3 and SW626 and ovarian epithelial cell line IOSE80 were treated with different concentrations of FXT(0,10,20,40,60,80 and 100 μmol/L),and the cytotoxicity of FXT was detected by methylthiazolyl diphenyl-tetrazolium bromide(MTT)assay.SKOV3 and SW626 cells were randomly divided into control group,FXT low,medium,high dose(40,60,80 μmol/L)group and FXT(80 μmol/L)+colivelin(STAT3 agonist,0.5 μmol/L)group.Colony formation assay and Transwell assay were used to detect cell proliferation,invasion and migration.The expressions of epithelial-mesenchymal transition and TLR4/STAT3 pathway related proteins were measured by Western blot.Results Compared with IOSE80 cells,SKOV3 and SW626 cell survival gradually decreased with increasing FXT concentration,and the differences were statistically significant(F=134.283,146.831,P<0.001).Compared with the control group,the relative colony lineage rates of SKOV3 and SW626 cells in the FXT low-,medium-and high-dose groups,invasion rate and mobility were reduced(t=4.433～45.909),E-cadherin were increased(t=5.879～17.345),and the expression of N-cadherin,zinc finger transcription factors(snail),vimentin,TLR4,phosphorylated(p)-STAT3/STAT3,cell cycle protein D1(Cyclin D1)and MYC oncogenes(Cancer-myc,C-myc)expression were sequentially reduced(t=7.348～50.117),and the differences were statistically significant(all P<0.01),respectively.Compared with the high-dose FXT group,the relative colony formation rate,cell invasion rate and migration rate of SKOV3 and SW626 cells in the FXT+colivelin group increased(t=9.224～20.703),while the expression of E-cadherin protein decreased(t=3.104,5.041),the expression of N-cadherin,snail,vimentin,TLR4,p-STAT3/STAT3,Cyclin D1,and C-myc increased(t=8.403～42.175),and the differences were statistically significant(all P<0.05),respectively.Conclusion FXT may exert antitumor effects by antagonizing the activation of TLR4/STAT3 signaling pathway and inhibiting ovarian cancer cell proliferation,migration,invasion and epithelial mesenchymal transition.

The Chinese Pharmacopoeia 2025 Edition added the 9213 Guideline for validation,verification,and transfer of microbiological analytical methods.Based on the characteristics of pharmaceutical microbiological analyt-ical methods and practical applications,it specified definitions of relevant terms and application scenarios,estab-lished technical indicators and acceptance criteria for methodological evaluation,and introduced key statistical tools and evaluation principles.This article systematically elaborates on the drafting background and process of the Guideline,and interprets its key content,aiming to offer theoretical guidance and practical reference for relevant practitioners in applying this guideline.This guideline strengthens the foundation of pharmaceutical microbial analytical methods in China and enhances the scientificity and accuracy of the pharmaceutical microbial standards system.

The Chinese Pharmacopoeia 2025 Edition added the 9213 Guideline for validation,verification,and transfer of microbiological analytical methods.Based on the characteristics of pharmaceutical microbiological analyt-ical methods and practical applications,it specified definitions of relevant terms and application scenarios,estab-lished technical indicators and acceptance criteria for methodological evaluation,and introduced key statistical tools and evaluation principles.This article systematically elaborates on the drafting background and process of the Guideline,and interprets its key content,aiming to offer theoretical guidance and practical reference for relevant practitioners in applying this guideline.This guideline strengthens the foundation of pharmaceutical microbial analytical methods in China and enhances the scientificity and accuracy of the pharmaceutical microbial standards system.

Objective To investigate the effect of fraxetin(FXT)on the malignant biological behavior of ovarian cancer cells by regulating Toll-like receptor 4(TLR4)/signal transducer and activator of transcription 3(STAT3)pathway.Methods Ovarian cancer cell lines SKOV3 and SW626 and ovarian epithelial cell line IOSE80 were treated with different concentrations of FXT(0,10,20,40,60,80 and 100 μmol/L),and the cytotoxicity of FXT was detected by methylthiazolyl diphenyl-tetrazolium bromide(MTT)assay.SKOV3 and SW626 cells were randomly divided into control group,FXT low,medium,high dose(40,60,80 μmol/L)group and FXT(80 μmol/L)+colivelin(STAT3 agonist,0.5 μmol/L)group.Colony formation assay and Transwell assay were used to detect cell proliferation,invasion and migration.The expressions of epithelial-mesenchymal transition and TLR4/STAT3 pathway related proteins were measured by Western blot.Results Compared with IOSE80 cells,SKOV3 and SW626 cell survival gradually decreased with increasing FXT concentration,and the differences were statistically significant(F=134.283,146.831,P<0.001).Compared with the control group,the relative colony lineage rates of SKOV3 and SW626 cells in the FXT low-,medium-and high-dose groups,invasion rate and mobility were reduced(t=4.433～45.909),E-cadherin were increased(t=5.879～17.345),and the expression of N-cadherin,zinc finger transcription factors(snail),vimentin,TLR4,phosphorylated(p)-STAT3/STAT3,cell cycle protein D1(Cyclin D1)and MYC oncogenes(Cancer-myc,C-myc)expression were sequentially reduced(t=7.348～50.117),and the differences were statistically significant(all P<0.01),respectively.Compared with the high-dose FXT group,the relative colony formation rate,cell invasion rate and migration rate of SKOV3 and SW626 cells in the FXT+colivelin group increased(t=9.224～20.703),while the expression of E-cadherin protein decreased(t=3.104,5.041),the expression of N-cadherin,snail,vimentin,TLR4,p-STAT3/STAT3,Cyclin D1,and C-myc increased(t=8.403～42.175),and the differences were statistically significant(all P<0.05),respectively.Conclusion FXT may exert antitumor effects by antagonizing the activation of TLR4/STAT3 signaling pathway and inhibiting ovarian cancer cell proliferation,migration,invasion and epithelial mesenchymal transition.

Objective To observe the clinical efficacy of Shen Shuai Ⅱ Granules combined with integrated Western medicine therapy in patients with primary chronic kidney disease(CKD)in stages 3-4 with the syndrome of spleen and kidney qi deficiency,dampness turbidity and blood stasis,and its effects on expression of serum autophagy related proteins.Methods Totally 88 patients were randomly divided into treatment group and control group,with 44 cases in each group.Both groups were given an integrated Western medicine therapy.On this basis,the treatment group was given Shen Shuai Ⅱ Granules,while the control group was given Shen Shuai Ⅱ placebo,1 bag at a time,twice a day,orally.The treatment for both groups lasted for 24 weeks.The clinical efficacy of the two groups was observed.The TCM symptom scores,serum creatinine(Scr),blood urea nitrogen(BUN),uric acid(UA),blood calcium(Ca2+),blood phosphorus(P3-),parathyroid hormone(PTH),and 24-hour urinary protein quantification(24 UPro)before and after treatment were compared in the two groups.The serum levels of autophagy related protein Beclin-1,Atg7,and LC3-Ⅱ were detected by ELISA before and after treatment in the two groups.Safety indexes of both groups were monitored.Results During the treatment,4 cases fell off in the treatment group and 4 cases in the control group.The total effective rate in the treatment group was 55％(22/40),while that in the control group was 30％(12/40).The therapeutic effect in the treatment group was better than that in the control group(P<0.01).Compared with before treatment,the TCM symptom scores in the treatment group decreased after treatment(P<0.01),the levels of Scr,BUN,UA,P3-,PTH,and 24 UPro decreased(P<0.05,P<0.01),while eGFR increased(P<0.01).The levels of Scr in the control group increased(P<0.05),while eGFR decreased(P<0.01).After treatment,the treatment group had lower various TCM symptom scores,Scr,BUN,UA,P3-,PTH,and 24 UPro levels than the control group(P<0.01,P<0.05),and higher eGFR than the control group(P<0.01).Compared with before treatment,the serum levels of Beclin-1,Atg7,and LC3-Ⅱ increased in the treatment group after treatment(P<0.01,P<0.05),while the serum level of Beclin-1 decreased in the control group after treatment(P<0.01).After treatment,the improvement of the above indicators in the treatment group were better than those in the control group(P<0.01,P<0.05).Correlation analysis showed that Beclin-1,Atg7,and LC3-Ⅱ were negatively correlated with Scr and BUN(P<0.05),and positively correlated with eGFR(P<0.05).There was no obvious adverse reaction in both groups.Conclusion The combination of Shen Shuai Ⅱ Granules and integrated Western medicine therapy for the treatment of primary CKD in stages 3-4 with the syndrome of spleen and kidney qi deficiency,dampness turbidity and blood stasis has confirmed efficacy,which can effectively alleviate clinical symptoms,improve renal function,and delay the progression of CKD.The mechanism may be related to improving the level of autophagy in renal.

Objective·To systematically integrate relevant influencing factors of advanced cancer patients'engagement behavior in advance care planning(ACP).Methods·The systematic search of Chinese and English literature on factors influencing ACP engagement behavior in advanced cancer patients from inception to December 2022 in China National Knowledge Infrastructure(CNKI),Wanfang,China Biomedical Literature Database(Sinomed),PubMed,Cochrane Library,Embase,CINAHL,and PsycINFO was conducted.After the literature quality evaluation,content extraction and summary were conducted by two researchers,and the data of quantitative research and qualitative research were extracted and integrated respectively.The final influencing factors of ACP engagement behavior of advanced cancer patients were obtained.With the help of the theoretical domain,they were mapped to the capability,opportunity,motivation-behavior(COM-B)model step by step.Results·A total of 21 studies were included and 27 factors were summarized,including 9 theoretical domains.Mapping to the COM-B model included 9 capability factors(knowledge of ACP,education level,accurate knowledge of prognosis,knowledge of the time of disease diagnosis,prior experience,subjective life expectancy,age,cancer site,and disease symptom burden),13 opportunity factors(gender,marital status,race/ethnicity,religious belief,dependent children,family economic condition,place of living,housing type,family support,social support,doctor-patient relationship,acculturation,and whether or not to establish a hospice service center)and 5 motivational factors(ACP attitude,ACP belief,ACP motivation,anxiety and depression,and death attitude).Among them,doctor-patient relationship,religious belief,ACP attitude,educational level,marital status,family support,knowledge of ACP,accurate knowledge of prognosis,age,place of living,attitude toward death,prior experience,and race/ethnicity were more influential factors on ACP engagement behavior.Conclusion·Based on the COM-B model,the influencing factors of ACP engagement behavior in advanced cancer patients can be comprehensively summarized.Future studies can use the above factors as an entry point to design continuous,multifaceted,and comprehensive interventions based on the COM-B model to promote the practice of ACP engagement behavior in advanced cancer patients.

Objective To observe the effect on clinical efficacy and renal microcirculation(RMC)of Shenshuai Ⅱ Prescription Granules in the treatment of patients with stage 3-4 chronic kidney disease(CKD)caused by primary glomerulonephritis(PGN)with deficiency of spleen and kidney qi syndrome.Methods Using a randomized,double-blind clinical trial design,70 patients with CKD stage 3-4 were divided into the treatment group and the control group,with 35 cases in each group.The control group was given CKD integrated treatment combined with placebo granules,and the treatment group was given CKD integrated treatment combined with Shenshuai Ⅱ Prescription Granules.The clinical efficacy of both groups was observed.TCM symptom scores,renal function indexes and RMC indexes before and after treatment of the two groups were compared.Safety indexes of both groups were monitored.Results The total effective rate was 82.86％(29/35)in the treatment group and 54.29％(19/35)in the control group,with statistical significance(P<0.05).Compared with before treatment,all the TCM symptom scores of the 2 groups were reduced after treatment(P<0.05,P<0.01);after treatment,the 5 spleen and kidney qi deficiency symptom scores and the total score of the treatment group were lower than those of the control group(P<0.05).The mean values of renal cortical and medullary blood flow were elevated in the treatment group after treatment compared with the pre-treatment period in this group(P<0.05,P<0.01);the mean values of renal cortical and medullary blood flow were elevated in the treatment group compared with the control group in the post-treatment period(P<0.05,P<0.01).Compared with before treatment,the serum content of NO increased(P<0.01),ET-1 and VEGF decreased(P<0.01);after treatment,the serum content of NO in the treatment group was higher than that in the control group(P<0.05),and ET-1 and VEGF in the treatment group were lower than those in the control group(P<0.01).Compared with before treatment,SCr,BUN,24 hUP decreased and eGFR increased in the treatment group(P<0.05,P<0.01);after treatment,SCr,BUN,and 24 hUP in the treatment group were lower than those in the control group,and eGFR in the treatment group was higher than that in the control group(P<0.01).Compared with this group before treatment and between groups after treatment,the difference in Uric acid between the 2 groups was not statistically significant(P>0.05).There was no abnormal change in safety indexes before and after treatment in both groups(P>0.05).Conclusion Shenshuai Ⅱ Prescription Granules combined with CKD integrated treatment can effectively improve clinical symptoms and renal function related indexes of CKD stage 3-4 patients with spleen and kidney qi deficiency syndrome,increase renal blood flow,improve RMC disorder,and improve clinical efficacy.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

Objective:To compare the differences in the prevalence of mild micro-hepatic encephalopathy (MHE) among patients with cirrhosis by using the psychometric hepatic encephalopathy score (PHES) and the Stroop smartphone application (Encephal App) test.Methods:This prospective, multi-center, real-world study was initiated by the National Clinical Medical Research Center for Infectious Diseases and the Portal Hypertension Alliance and registered with International ClinicalTrials.gov (NCT05140837). 354 cases of cirrhosis were enrolled in 19 hospitals across the country. PHES (including digital connection tests A and B, digital symbol tests, trajectory drawing tests, and serial management tests) and the Stroop test were conducted in all of them. PHES was differentiated using standard diagnostic criteria established by the two studies in China and South Korea. The Stroop test was evaluated based on the criteria of the research and development team. The impact of different diagnostic standards or methods on the incidence of MHE in patients with cirrhosis was analyzed. Data between groups were differentiated using the t-test, Mann-Whitney U test, and χ2 test. A kappa test was used to compare the consistency between groups. Results:After PHES, the prevalence of MHE among 354 cases of cirrhosis was 78.53% and 15.25%, respectively, based on Chinese research standards and Korean research normal value standards. However, the prevalence of MHE was 56.78% based on the Stroop test, and the differences in pairwise comparisons among the three groups were statistically significant (kappa = -0.064, P < 0.001). Stratified analysis revealed that the MHE prevalence in three groups of patients with Child-Pugh classes A, B, and C was 74.14%, 83.33%, and 88.24%, respectively, according to the normal value standards of Chinese researchers, while the MHE prevalence rates in three groups of patients with Child-Pugh classes A, B, and C were 8.29%, 23.53%, and 38.24%, respectively, according to the normal value standards of Korean researchers. Furthermore, the prevalence rates of MHE in the three groups of patients with Child-Pugh grades A, B, and C were 52.68%, 58.82%, and 73.53%, respectively, according to the Stroop test standard. However, among the results of each diagnostic standard, the prevalence of MHE showed an increasing trend with an increasing Child-Pugh grade. Further comparison demonstrated that the scores obtained by the number connection test A and the number symbol test were consistent according to the normal value standards of the two studies in China and South Korea ( Z = -0.982, -1.702; P = 0.326, 0.089), while the other three sub-tests had significant differences ( P < 0.001). Conclusion:The prevalence rate of MHE in the cirrhotic population is high, but the prevalence of MHE obtained by using different diagnostic criteria or methods varies greatly. Therefore, in line with the current changes in demographics and disease spectrum, it is necessary to enroll a larger sample size of a healthy population as a control. Moreover, the establishment of more reliable diagnostic scoring criteria will serve as a basis for obtaining accurate MHE incidence and formulating diagnosis and treatment strategies in cirrhotic populations.

Abstract OBJECTIVE To develop an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for determination of hynchophylline, isorhynchophylline, isocorynoxeine, corynoxeine, hirsutine and hirsuteine in Uncaria decoction, and investigate the effect of different storage conditions on the stability of active ingredients. METHODS An UHPLC-MS/MS method was established to investigate the effects of different storage temperature and time on the stability of six active components in Uncaria decoction. Separation was performed with a gradient mobile phase system of acetonitrile-0.1% formic water solution, the flow rate was 0.4 mL·min-1, the temperature of column was 30℃, the injection volume was 2 μL, the MS detection was dynamic multiple reaction monitoring mode. The effects of different storage temperature and time on the stability of six active components in Uncaria decoction was investigated. RESULTS Rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine and hirsuteine were successfully separated using this method, with good linear relationship in the corresponding concentration range. The precision, repeatability, stability and recovery rate were good. The six alkaloids were basically stable within 14 d under low temperature storage, basically stable within 3 d under normal temperature storage, and not stable under high temperature storage. CONCLUSION The UHPLC-MS/MS method is stable, rapid and reproducible. It can be used to determine the contents of six effective components in Uncaria decoction. Cold storage is beneficial to the stability of Uncaria decoction.