OBJECTIVE To establish an UHPLC-MS method for simultaneous determination of 9 bile acids components in Shedan Chuanbei liquid. To analyze the differences of bile acids components from different manufacturers and different batches of product. METHODS Thermo Fisher Scientfic Bremen HYPERSIL GOLD(2.1 mm×100 mm, 1.9 µm) was selected as the chromatographic column. The mobile phase was 0.1% formic acid-water and methanol with gradient elution at a flow rate of 0.2 mL·min−1, and the column temperature was 40 ℃. HESI negative ion mode was used for mass spectrometry, and PRM scanning mode was used for mass spectrometry data. RESULTS The calibration curves of 9 biles acids were linear in their own range(r≥0.999 2). The average recoveries ranged from (95.5±1.52)% to (103.1±3.81)%. The RSD ranged from 1.1% to 5.5%. Nine bile acids components including taurocholic acid, tauroursodesoxycholic acid, 7-ketone-3α,12-α-hydroxyl cholanic acid, taurochenodeoxy-cholic acid, sodium taurodeoxycholate, cholic acid, sodium taurocholate, chenodeoxycholic acid and deoxycholic acid were tentatively identified from 19 batches of test samples. Taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate sodium were detected in all the test batches. CONCLUSION This method is simple and stable, and has certain reference value for improving the quality control and evaluation of Shedan Chuanbei liquid.

Objective: To investigate the prevalence of frailty and its influencing factors among the elderly, so as to provide the evidence for prevention and control of frailty. Methods: The elderly population at ages of 65 years and older were selected from 14 administrative villages or communities in Wuzhong District, Suzhou City, Jiangsu Province, using the random cluster sample method from July to November, 2022. Demographic information, smoking and alcohol consumption were collected through questionnaire surveys. Physical activity, sleep quality and frailty were evaluated using the International Physical Activity Questionnaire-Short, Pittsburgh Sleep Quality Index and the FRAIL Scale, respectively. Factors affecting frailty among the elderly were evaluated using a multinomial logistic regression model. Results: A total of 8 782 elderly peolple were surveyed, including 4 259 males (48.50%) and 4 523 females (51.50%). The median age was 71.00 (interquartile range, 8.00) years. There were 2 145 cases with pre-frailty (24.42%) and 189 cases with frailty (2.15%). Multinomial logistic regression analysis showed that age (75-<85 years, OR=1.330, 95%CI: 1.186-1.492; ≥85 years, OR=2.452, 95%CI: 1.882-3.195), smoking (current smoking, OR=0.838, 95%CI: 0.714-0.983), physical activity level (low, OR=1.161, 95%CI: 1.010-1.333) and sleep quality (poor, OR=2.248, 95%CI: 1.822-2.774) were associated with pre-frailty; age (75-<85 years,OR=2.629, 95%CI: 1.921-3.596; ≥85 years, OR=3.067, 95%CI: 1.621-5.801), educational level (junior high school and above, OR=0.488, 95%CI: 0.298-0.798), body mass index (underweight, OR=1.848, 95%CI: 1.023-3.337; obesity, OR=1.798, 95%CI: 1.180-2.740), smoking (quit smoking, OR=1.787, 95%CI: 1.007-3.171; current smoking, OR=0.448, 95%CI: 0.242-0.830), alcohol consumption (yes, OR=0.532, 95%CI: 0.291-0.972), physical activity level (low, OR=2.757, 95%CI: 1.646-4.616) and sleep quality (poor, OR=3.911, 95%CI: 2.438-6.273) were associated with frailty. Conclusion Older, low physical activity level, poor sleep quality, underweight and obesity are associated with frailty of the elderly.

Cancer stands as one of the predominant causes of mortality globally, necessitating ongoing efforts to develop innovative therapeutics. Historically, natural products have been foundational in the quest for anticancer agents. Bulbocodin D (BD) and Bulbocodin C (BC), two bibenzyls derived from Pleione bulbocodioides (Franch.) Rolfe, have demonstrated notable in vitro anticancer activity. In human lung cancer A549 cells, the IC_50s for BD and BC were 11.63 and 11.71 μmol·L^-1, respectively. BD triggered apoptosis, as evidenced by an upsurge in Annexin V-positive cells and elevated protein expression of cleaved-PARP in cancer cells. Furthermore, BD and BC markedly inhibited the migratory and invasive potentials of A549 cells. The altered genes identified through RNA-sequencing analysis were integrated into the CMap dataset, suggesting BD's role as a potential signal transducer and activator of transcription 3 (STAT3) inhibitor. SwissDock and MOE analyses further revealed that both BD and BC exhibited a commendable binding affinity with STAT3. Additionally, a surface plasmon resonance assay confirmed the direct binding affinity between these compounds and STAT3. Notably, treatment with either BD or BC led to a significant reduction in p-STAT3 (Tyr 705) protein levels, regardless of interleukin-6 stimulation in A549 cells. In addition, the extracellular signal-regulated kinase (ERK) was activated after BD or BC treatment. An enhancement in cancer cell mortality was observed upon combined treatment of BD and U0126, the MEK1/2 inhibitor. In conclusion, BD and BC emerge as promising novel STAT3 inhibitors with potential implications in cancer therapy.
Humans ; Lung Neoplasms/metabolism* ; STAT3 Transcription Factor/metabolism* ; Antineoplastic Agents/chemistry* ; A549 Cells ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation

ObjectiveTo explore the effect of Babaodan （BBD） on the NOD-like receptor pyrin domain containing 3/cysteine aspartate-specific protease-3 （NLRP3/Caspase-1） pathway proteins in mice with acetaminophen （APAP）-induced acute liver injury. MethodC57BL/6 mice were randomly grouped， and BBD （75， 150， 300 mg·kg^-1， ig） was administered twice a day for three days. After 2 hours of the last administration， the mice were treated with APAP （400 mg·kg^-1， ip）， and the eyeballs were removed to collect blood after 14 hours. Then they were sacrificed by cervical dislocation for sample collection. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of liver tissue cells， and biochemical methods were used to detect the activities of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， superoxide dismutase （SOD）， malondialdehyde （MDA） and myeloperoxidase （MPO） in serum of mice in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was performed to determine the mRNA expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β） and IL-6， and Western blot was performed to determine the protein expression of cyclooxygenase-2 （COX-2）， inducible nitric oxide synthase （iNOS）， NLRP3， Caspase-1 and IL-18 in the liver of mice. ResultCompared with the conditions in normal group， the hepatic lobule structure of mice in the model group was partially destroyed， and the hepatic sinusoids were dilated. And the expression levels of ALT and AST in serum， the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the mRNA levels of IL-1β， IL-6 and TNF-α were increased （P<0.05， P<0.01）. Compared with the model group， the administration groups had improvement in liver cell rupture and hepatic sinusoidal compression， and a dose-dependent decrease in the levels of ALT and AST in serum as well as the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the the mRNA levels of IL-1β， IL-6 and TNF-α in liver tissue （P<0.05， P<0.01）. ConclusionBBD can reduce APAP-induced acute liver injury in mice. The mechanism may be related to anti-oxidative stress， inhibition of NLRP3/Caspase-1 pathway， and decreased expression levels of IL-1β， IL-18， TNF-α and IL-6.

ObjectiveTo investigate the principle and scientific connotation of Euodiae Fructus（EF） processed with Glycyrrhizae Radix et Rhizoma（Gly） by comparing the effects of unprocessed products of EF（UEF） and processed products of EF with the different proportions of Gly（GEFs） at toxic doses on oxidative stress and autophagy in the liver of mice. MethodSeventy mice were randomly divided into 7 groups， namely the control group， the UEF group， the group of the processed products of EF without Gly（PEF） and 4 groups of GEFs（the mass ratios of EF to Gly were 100∶3， 100∶6， 100∶12 and 100∶24， respectively， hereinafter referred to as the processed products of EF with the mass ratios of 100∶3， 100∶6， 100∶12 and 100∶24 of Gly）. The mice were given purified water， the decoction of UEF， PEF and GEFs by gavage at a dose of 30 g·kg^-1. PEF and GEFs were prepared according to the method under EF in the 2020 edition of Chinese Pharmacopoeia. Levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were determined by ultraviolet-visible spectrophotometry， hematoxylin-eosin（HE） staining was used to evaluate the pathological changes of liver tissue， the level of reactive oxygen species（ROS） was detected by fluorescence method， the mRNA expression of heme oxygenase-1（HO-1）， quinone oxidoreductase-1（NQO1）， glutathione-S-transferase 1（GSTA1）， Kelch-like epichlorohydrin-associated protein 1（Keap1） and p62 were measured by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， western blot was used to detect the protein expression of phosphorylated mammal target of rapamycin（p-mTOR）， phosphorylated ribosomal p70 S6 protein kinase（p-p70S6K）， p62， microtubule-associated protein 1 light chain 3Ⅰ（LC3Ⅰ） and LC3Ⅱ. ResultCompared with the control group， after 7 d of administration， the increase in body mass of mice in the UEF group began to slow down and the difference gradually increased， and the liver body index significantly increased（P<0.01）， pathomorphological observation showed that the structure of hepatic lobules was disordered， and local hepatic sinuses were narrowed or disappeared， and there were inflammatory infiltration and local bleeding， the levels of ALT and AST in serum and ROS in liver tissue were significantly increased（P<0.01）， and the expressions of Keap1， HO-1， NQO1， GSTA1， p62 mRNA and p-mTOR， p-p70S6K， p62 protein in liver tissue were significantly decreased（P<0.01）， and LC3Ⅱ/LC3Ⅰ was significantly increased（P<0.01）. Compared with the UEF group， the body mass of mice increased， and the liver body index， the levels of ALT and AST in serum， and the level of ROS in liver tissue all decreased in the groups of PEF and GEFs. Among these groups， only the liver lobules in GEF（100∶6） group were intact， and the size of liver sinuses was close to that in the control group. The mRNA expressions of Keap1， HO-1， NQO1， GSTA1 and p62 in liver tissue showed an overall upward trend in the groups of PEF and GEFs. Among these groups， only the ones of the above mRNA in the GEF（100∶6） group had a significant increase（P<0.05， P<0.01）. The protein expressions of p-mTOR， p-p70S6K， p62 and LC3Ⅱ/LC3Ⅰ had a callback in the groups of PEF and GEFs， of which the protein expressions of p-mTOR， p-p70S6K and LC3Ⅱ/LC3Ⅰ in the GEF（100∶6） group and the expression of p62 protein in the GEF（100∶24） group had the largest callback. Except for p-mTOR protein， other protein expressions were statistically significant（P<0.05， P<0.01）. ConclusionThe hepatotoxicity of EF is closely related to its ability to induce oxidative stress， which leads to pathological autophagy and hepatocyte damage. This ability can be reduced by the processing with different proportions of Gly， especially the ratio of 100∶6.

OBJECTIVE To establish a method for simultaneous determination of 15 bile acids in Tongren niuhuang qingxin pills， and to determine the contents of 15 batches of samples. METHODS Using dehydrocholic acid as internal standard， the determination was performed by ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. The determination was performed on Hypersil GOLD C18 column with methanol-0.1% formic acid solution as the mobile phase by gradient elution at the flow rate of 0.2 mL/min. The column temperature was 40 ℃ ， and the sample size was 2 µL. Using heated electrospray ion source， parallel reaction monitoring mode scanning was performed in negative ion mode. SPSS 24.0 software was used for chemical pattern recognition analysis of content determination results. RESULTS The 15 bile acid components had a good linear relationship with peak area （all R2≥0.998 9）； their precision， repeatability and stability were all good （all RSD≤5.49%）； the average recoveries were 93.8%-105.7% （RSD was 0.5%-5.8%）. The average contents of taurocholic acid， 7-oxodeoxycholic acid， 12-dehydrocholic acid， glycocholic acid， 3-oxo-7α， 12α-hydroxy-5β-cholanoic acid， taurochenodeoxycholic acid， 3α-hydroxy- 7-oxo-5β -cholanic acid， hyocholic acid， taurodeoxycholic acid sodium salt hydrate， hyodeoxycholic acid， cholic acid， glycochenodeoxycholic acid， glycodeoxycholic acid， chenodeoxycholic acid and deoxycholic acid were 670.56， 25.97， 10.54， 280.12， 4.04， 29.81， 182.98， 813.55， 120.95， 220.31， 797.37， 18.37， 68.59， 30.13， 59.82 μg/g， respectively. Both cluster analysis and principal component analysis divided 15 batches of Tongren niuhuang qingxin pills into 2 categories， S1-S12 as one category and S13-S15 as the other category. CONCLUSIONS The established method is accurate， sensitive and specific， and can determine many types of bile acids. It also can quickly achieve the quantitative analysis of 15 bile acids in Tongren niuhuang qingxin pills， which is suitable for the quality control of this drug.

OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites， theophylline， paraxanthine and theobromine in urine， and apply it in clinical practice. METHODS Using caffeine-13C3-d3 as internal standard （IS）， and the urine samples were protein precipitated with acetonitrile； HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites. The determination was performed on Waters ACQUITY UPLC® BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate （A）-acetonitrile （B） （gradient elution） at the flow rate of 0.5 mL/min. The column temperature was set at 38 ℃ ， and the sample size was 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine， m/z 181.1→124.0 for theophylline， m/z 181.1→124.0 for paraxanthine， m/z 181.1→138.0 for theobromine， and m/z 198.1→ 140.1 for IS. The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity （AOP）. RESULTS The linear ranges of mass concentration of caffeine， theophylline， paraxanthin and theobromine were 0.200-200， 0.050-50.0，0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （r＞0.990）， respectively. RSDs of intra-day and intra- day precision were not above 10.37%， and matrix factors were 85.68%-109.90%； extraction recoveries were 93.53%-109.40% （RSD≤15%）， and RSDs of stability tests were all lower than 15%. The concentrations of caffeine and its three metabolites in the urine of 19 cases were （27.346±7.951）， （0.351±0.223）， （0.428±0.395） and （0.472±0.374） μg/mL， respectively. CONCLUSIONS The established HPLC-MS/MS method is simple， sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.

ObjectiveTo explain the scientific connotation of Morindae Officinalis Radix （MOR） processed by Glycyrrhizae Radix et Rhizoma （Gly） by comparing the effect of raw products of MOR and processed products of MOR with different proportions of Gly （GMOs） on the improvement of renal function and hypothalamic-pituitary-gonadal （HPG） axis， the protein expression of Wnt/β-catenin and transforming growth factor-β₁ （TGF-β₁）/Smad signal pathways in kidney Yang deficiency model rats induced by adenine. MethodGMOs were prepared according to method under MOR in 2020 edition of Chinese Pharmacopoeia. Rat model of kidney Yang deficiency was established by intragastrical administration of adenine， levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E₂） and testosterone （T） were measured by enzyme-linked immunosorbent assay （ELISA）. Levels of urea nitrogen （BUN） and serum creatinine （SCr） were measured by spectrophotometry， hematoxylin-eosin （HE） staining was used to evaluate the pathological changes of kidney， testis and epididymis. Immunohistochemistry （IHC） was used to analyze the protein expression of E-cadherin， α-smooth muscle actin （α-SMA）， Wnt2b， β-catenin， Smad1 and Smad4. ResultMOR processed with 100∶6 and 100∶12 proportions of Gly （short for GMO/100∶6 and GMO/100∶12） had the most obvious improvement on the body posture of kidney Yang deficiency model rats. GMO/100∶12 had the best effect on reducing the levels of BUN， SCr， FSH， LH and the ratio of E₂/T. GMO/100∶6 and GMO/100∶12 had the best effect on regulating the protein expression of E-cadherin， α-SMA， Wnt2b， β-catenin， Smad1 and Smad4. ConclusionGMO/100∶6 and GMO/100∶12 have the a good effect on the improvement of renal function and HPG axis in kidney Yang deficiency model rats induced by adenine， which is related with the fact that they can regulate Wnt/β-catenin pathway in renal and testicular tissue and TGF-β₁/Smads pathway in testicular tissue.

Clinical data of 43 patients who underwent endoscopic resection for gastrointestinal stromal tumors (GIST) of length ≤1.2 cm at the Digestive Endoscopy Center of the 909th Hospital from January 2016 to December 2018 were retrospectively analyzed. The patients were divided into the endoscopic ligation resection (ELR) group ( n=27) and the endoscopic submucosal excavation (ESE) group ( n=16). The general, perioperative and follow-up data of the two groups were compared. The results showed that there was no significant difference in the general data between the two groups. The operation time was 20.0 (18.0,25.0) min in the ELR group and 27.5 (23.0,37.5) min in the ESE group, showing significant difference ( U=92.5, P=0.001). The en bloc resection rates were 100.0% (27/27) in the ELR group and 81.3% (13/16) in the ESE group, showing significant difference ( P=0.045). The postoperative hospital stays were 3 (2,4) days in the ELR group and 5 (4,6) days in the ESE group, showing significant difference ( U=125.5, P=0.020). There was no significant difference in the intraoperative bleeding rate, intraoperative hemorrhage volume, intraoperative perforation rate, number of hemostatic clips or postoperative complications including hemorrhage, fever and peritonitis between the two groups ( P>0.05). During the follow-up, there was no recurrence or metastasis of GIST in both groups. ELR and ESE can be safe and effective for small GIST ≤1.2 cm in diameter. Compared with the ESE group, the operation time and postoperative hospital stay are shorter with higher en bloc resection rate in the ELR group.

Objective:To establish autoverification rules for coagulation tests in multicenter cooperative units, in order to reduce workload for manual review of suspected results and shorten turnaround time (TAT) of test reports, while ensure the accuracy of results.Methods:A total of 14 394 blood samples were collected from fourteen hospitals during December 2019 to March 2020. These samples included: Rules Establishment Group 11 230 cases, including 1 182 cases for Delta check rules; Rules Validation Group 3 164 cases, including 487cases for Delta check; Clinical Application Trial Group 77 269 cases. Samples were analyzed for coagulation tests using Sysmex CS series automatic coagulation analyzers, and the clinical information, instrument parameters, test results, clinical diagnosis, medication history of anticoagulant and other relative results such as HCT, TG, TBIL, DBIL were summarized; on the basis of historical data, the 2.5 and 97.5 percentile of all data arranged from low to high were initially accumulated; on the basis of clinical suggestions, critical values and specific drug use as well as relative guidelines, autoverification rules and limits were established.The rules were then input into middleware, in which Stage I/Stage II validation was done. Positive coincidence, negative coincidence, false negative, false positive, autoverification pass rate, passing accuracy (coincidence of autoverification and manual verification) were calculated. Autoverification rules underwent trial application in coagulation results reports.Results:(1) The autoverification algorisms involve 33 rules regarding PT/INR, APTT, FBG, D-dimer, FDP,Delta check, reaction curve and sample abnormalities; (2)Autoverification Establishment Group showed autoverification pass rate was 68.42% (7 684/11 230), the false negative rate was 0%(0/11230), coincidence of autoverification and manual verification was 98.51%(11 063/11 230), in which positive coincidence and negative coincidence were respectively 30.09% (3 379/11 230) and 68.42%(7 684/11 230); Autoverification Validation Group showed autoverification pass rate was 60.37%(1 910/3 164), the false negative rate was 0%(0/11 230), coincidence of autoverification and manual verification was 97.79%(3 094/3 164), in which positive coincidence and negative coincidence were respectively 37.42%(1 184/3 164) and 60.37%(1 910/3 164); (3) Trialed implementation of these autoverification rules on 77 269 coagulation samples showed that the average TAT shortened by 8.5 min-83.1 min.Conclusions:This study established 33 autoverification rules in coagulation tests. Validation showedthese rules could ensure test quality while shortening TAT and lighten manual workload.