Damarane-type triterpene saponins are the main active ingredients in Gynostemma pentaphyllum (Thunb.) Makino. By using D101 macroporous adsorption resin and silica gel open-columns, C18 medium-pressure column chromatography, and preparative high performance liquid chromatography, we isolated four compounds from the leaves of G. pentaphyllum planted in Guangxi Zhuang Autonomous Region. Their structures were determined by comprehensive analyses of MS, NMR data and circular dichroism spectroscopy and identified as (3β,12β)-dihydroxy-25-peroxyhydrohydrodammarane-20,23-diene-3-O-[β-D-glucopyranosyl(1→2)]-β-D-glucopyranoside (1), (3β,12β,24S)-trihydroxydammarane-20,25-diene-3-O-[β-D-glucopyranosyl(1→2)]-β-D-glucopyranoside (2), (3β,20α)-dihydroxy-24-en-12β,22S-epoxydammarane-3-O-[β-D-glucopyranosyl(1→2)]-β-D-glucopyranoside (3), (3β,12β,20S)-trihydroxydammarane-24-en-3-O-[6-O-acetyl-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl(1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (4). Compounds 1-4 are new dammarane-type triterpene saponins.

AIM To investigate the effects of Zhuangyao Shuanglu Tongnao Formula on neuronal apoptosis in rats with cerebral ischemia-reperfusion injury based on the study of oxidative stress and inflammatory response.METHODS The rats were randomly divided into the sham operation group,the model group,the edaravone group(3.0 mg/kg),the low,medium and high dose groups(9.0,18.0,36.0 g/kg)of Zhuangyao Shuanglu Tongnao Formula,with 18 rats in each group.The middle cerebral artery occlusion/reperfusion was conducted by thread embolism method to simulate cerebral ischemia reperfusion injury in rats followed by 6 days corresponding drugs administration.Subsequently,the rats had their neurological function deficit scored by Zeal Longa scoring method;their sizes of cerebral infarction areas measured by TTC staining;their pathological damage and apoptosis of neurons in hippocampal CA1 area of ischemic penumbra of the brain tissue detected by HE staining and TUNEL staining;their SOD activity and levels of GSH,MDA,IL-6,IL-1β,TNF-α in brain tissue detected by kits;and their protein expressions of Bax,Bcl-2,caspase-3,cleaved-capase-3,TLR4,NF-κB p65,Nrf2,HO-1 in rat brain tissue determined by Western blot.RESULTS Compared with the model group,the groups intervened with edaravone,medium and high dose of Zhuangyao Shuanglu Tongnao Formula displayed improvements in the scores of nerve function defects,the rate of cerebral infarction,the rate of neuronal apoptosis,the levels of IL-6,IL-1β,TNF-α and MDA in the ischemic penumbra of brain tissues,the protein expressions of Bax and TLR4,the ratio of cleaved-capase-3/caspase-3 and p-NF-κB p65/NF-κB p65(P＜0.05),the levels of GSH,the activity of SOD and the protein expressions of Bcl-2,Nrf2 and HO-1(P＜0.05).CONCLUSION Being an inhibitor of oxidative stress and inflammatory response,Zhuangyao Shuanglu Tongnao Formula can alleviate brain injury in rats with cerebral ischemia reperfusion injury through the inhibition of neuronal apoptosis and improvement of neural function mediated by the inhibition of TLR4/NF-κB signal pathway and activation of Nrf2/HO-1 signal pathway.

Background: and Purpose Intravenous tenecteplase (TNK) efficacy has not been well demonstrated in acute ischemic stroke (AIS) beyond 4.5 hours after onset. This study aimed to determine the effect of intravenous TNK for AIS within 4.5 to 24 hours of onset. Methods: In this pilot trial, eligible AIS patients with diffusion-weighted imaging (DWI)-fluid attenuated inversion recovery (FLAIR) mismatch were randomly allocated to intravenous TNK (0.25 mg/kg) or standard care within 4.5–24 hours of onset. The primary endpoint was excellent functional outcome at 90 days (modified Rankin Scale [mRS] score of 0–1). The primary safety endpoint was symptomatic intracranial hemorrhage (sICH). Results: Of the randomly assigned 80 patients, the primary endpoint occurred in 52.5% (21/40) of TNK group and 50.0% (20/40) of control group, with no significant difference (unadjusted odds ratio, 1.11; 95% confidence interval 0.46–2.66; P=0.82). More early neurological improvement occurred in TNK group than in control group (11 vs. 3, P=0.03), but no significant differences were found in other secondary endpoints, such as mRS 0–2 at 90 days, shift analysis of mRS at 90 days, and change in National Institutes of Health Stroke Scale score at 24 hours and 7 days. There were no cases of sICH in this trial; however, asymptomatic intracranial hemorrhage occurred in 3 of the 40 patients (7.5%) in the TNK group. Conclusion This phase 2, randomized, multicenter study suggests that intravenous TNK within 4.5–24 hours of onset may be safe and feasible in AIS patients with a DWI-FLAIR mismatch.

Objective: This cross-sectional investigation aimed to determine the incidence, clinical characteristics, prognosis, and related risk factors of olfactory and gustatory dysfunctions related to infection with the SARS-CoV-2 Omicron strain in mainland China. Methods: Data of patients with SARS-CoV-2 from December 28, 2022, to February 21, 2023, were collected through online and offline questionnaires from 45 tertiary hospitals and one center for disease control and prevention in mainland China. The questionnaire included demographic information, previous health history, smoking and alcohol drinking, SARS-CoV-2 vaccination, olfactory and gustatory function before and after infection, other symptoms after infection, as well as the duration and improvement of olfactory and gustatory dysfunction. The self-reported olfactory and gustatory functions of patients were evaluated using the Olfactory VAS scale and Gustatory VAS scale. Results: A total of 35 566 valid questionnaires were obtained, revealing a high incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain (67.75%). Females(χ2=367.013, P<0.001) and young people(χ2=120.210, P<0.001) were more likely to develop these dysfunctions. Gender(OR=1.564, 95%CI: 1.487-1.645), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), oral health status (OR=0.881, 95%CI: 0.839-0.926), smoking history (OR=1.152, 95%CI=1.080-1.229), and drinking history (OR=0.854, 95%CI: 0.785-0.928) were correlated with the occurrence of olfactory and taste dysfunctions related to SARS-CoV-2(above P<0.001). 44.62% (4 391/9 840) of the patients who had not recovered their sense of smell and taste also suffered from nasal congestion, runny nose, and 32.62% (3 210/9 840) suffered from dry mouth and sore throat. The improvement of olfactory and taste functions was correlated with the persistence of accompanying symptoms(χ2=10.873, P=0.001). The average score of olfactory and taste VAS scale was 8.41 and 8.51 respectively before SARS-CoV-2 infection, but decreased to3.69 and 4.29 respectively after SARS-CoV-2 infection, and recovered to 5.83and 6.55 respectively at the time of the survey. The median duration of olfactory and gustatory dysfunctions was 15 days and 12 days, respectively, with 0.5% (121/24 096) of patients experiencing these dysfunctions for more than 28 days. The overall self-reported improvement rate of smell and taste dysfunctions was 59.16% (14 256/24 096). Gender(OR=0.893, 95%CI: 0.839-0.951), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), history of head and facial trauma(OR=1.180, 95%CI: 1.036-1.344, P=0.013), nose (OR=1.104, 95%CI: 1.042-1.171, P=0.001) and oral (OR=1.162, 95%CI: 1.096-1.233) health status, smoking history(OR=0.765, 95%CI: 0.709-0.825), and the persistence of accompanying symptoms (OR=0.359, 95%CI: 0.332-0.388) were correlated with the recovery of olfactory and taste dysfunctions related to SARS-CoV-2 (above P<0.001 except for the indicated values). Conclusion: The incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain is high in mainland China, with females and young people more likely to develop these dysfunctions. Active and effective intervention measures may be required for cases that persist for a long time. The recovery of olfactory and taste functions is influenced by several factors, including gender, SARS-CoV-2 vaccination status, history of head and facial trauma, nasal and oral health status, smoking history, and persistence of accompanying symptoms.
Female ; Humans ; Adolescent ; SARS-CoV-2 ; Smell ; COVID-19/complications* ; Cross-Sectional Studies ; COVID-19 Vaccines ; Incidence ; Olfaction Disorders/etiology* ; Taste Disorders/etiology* ; Prognosis

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.
Cinnamomum camphora/enzymology* ; Alkyl and Aryl Transferases/chemistry*

OBJECTIVES: To compare the application value of the likelihood ratio (LR) method and identity by state (IBS) method in the identification involving half sibling relationships, and to provide a reference for the setting of relevant standards for identification of half sibling relationship. METHODS: (1) Based on the same genetic marker combinations, the reliability of computer simulation method was verified by comparing the distributions of cumulated identity by state score (CIBS) and combined full sibling index in actual cases with the distributions in simulated cases. (2) In different numbers of three genetic marker combinations, the simulation of full sibling, half sibling and unrelated individual pairs, each 1 million pairs, was obtained; the CIBS, as well as the corresponding types of cumulative LR parameters, were calculated. (3) The application value of LR method was compared with that of IBS method, by comparing the best system efficiency provided by LR method and IBS method when genetic markers in different amounts and of different types and accuracy were applied to distinguish the above three relational individual pairs. (4) According to the existing simulation data, the minimum number of genetic markers required to distinguish half siblings from the other two relationships using different types of genetic markers was estimated by curve fitting. RESULTS: (1) After the rank sum test, under the premise that the real relationship and the genetic marker combination tested were the same, there was no significant difference between the simulation method and the results obtained in the actual case. (2) In most cases, under the same conditions, the system effectiveness obtained by LR method was greater than that by IBS method. (3) According to the existing data, the number of genetic markers required for full-half siblings and half sibling identification could be obtained by curve fitting when the system effectiveness reached 0.95 or 0.99. CONCLUSIONS When distinguishing half sibling from full sibling pairs or unrelated pairs, it is recommended to give preference to the LR method, and estimate the required number of markers according to the identification types and the population data, to ensure the identification effect.
Humans ; Siblings ; Genetic Markers ; Computer Simulation ; Irritable Bowel Syndrome/genetics* ; Reproducibility of Results ; Genotype

OBJECTIVE: To investigate the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in regulating Bacillus Calmette-Guérin (BCG)-induced macrophage apoptosis. METHODS: The expression of TRAF6 in peripheral blood samples of 50 patients with active tuberculosis (TB) and 50 healthy individuals were detected using quantitative real-time PCR (qPCR). RAW264.7 macrophages were infected with BCG at different MOI and for different lengths of time, and the changes in expressions of Caspase 3 and TRAF6 were detected with Western blotting and qPCR. In a RAW264.7 cell model of BCG infection with TRAF6 knockdown established using RNA interference technique, the bacterial load was measured and cell apoptotic rate and mitochondrial membrane potential (MMP) were determined with flow cytometry. The expression levels of TRAF6, Caspase 3, PARP, BAX and Bcl-2 in the cells were detected using Western blotting, and the expressions of TRAF6 and Caspase 3 were also examined with immunofluorescence assay. RESULTS: The expression of TRAF6 was significantly upregulated in the peripheral blood of patients with active TB as compared with healthy subjects (P < 0.001). In RAW264.7 cells, BCG infection significantly increased the expressions of Caspase 3 and TRAF6, which were the highest in cells infected for 18 h and at the MOI of 15. TRAF6 knockdown caused a significant increase of bacterial load in BCG-infected macrophages (P=0.05), lowered the cell apoptotic rate (P < 0.001) and reduced the expressions of Caspase 3 (P=0.002) and PARP (P < 0.001). BCG-infected RAW264.7 cells showed a significantly increased MMP (P < 0.001), which was lowered by TRAF6 knockdown (P < 0.001); the cells with both TRAF6 knockdown and BCG infection showed a lowered BAX expression (P=0.005) and an increased expression of Bcl-2 (P=0.04). CONCLUSION TRAF6 promotes BCG-induced macrophage apoptosis by regulating the intrinsic apoptosis pathway.
Apoptosis ; BCG Vaccine ; Caspase 3/metabolism* ; Humans ; Intracellular Signaling Peptides and Proteins ; Macrophages ; Mycobacterium bovis/metabolism* ; Poly(ADP-ribose) Polymerase Inhibitors ; TNF Receptor-Associated Factor 6/metabolism* ; bcl-2-Associated X Protein/metabolism*

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.
Animals ; Epimedium/chemistry* ; Flavonoids/chemistry* ; Furans ; Glycosides/chemistry* ; Hydrolysis ; Male ; Molecular Docking Simulation ; Molecular Structure ; Rats ; Testosterone ; beta-Glucosidase/metabolism*

BACKGROUND: Shenyankangfu Tablet (SYKFT) is a Chinese patent medicine that has been used widely to decrease proteinuria and the progression of chronic kidney disease. OBJECTIVE: This trial compared the efficacy and safety of SYKFT, for the control of proteinuria in primary glomerulonephritis patients, against the standard drug, losartan potassium. DESIGN, SETTING, PARTICIPANTS AND INTERVENTION: This was a multicenter, double-blind, randomized, controlled clinical trial. Primary glomerulonephritis patients, aged 18-70 years, with blood pressure ≤ 140/90 mmHg, estimated glomerular filtration rate (eGFR) ≥ 45 mL/min per 1.73 m MAIN OUTCOME MEASURES: The primary outcome was change in the 24-hour proteinuria level, after 48 weeks of treatment. RESULTS: A total of 735 participants were enrolled. The percent decline of urine protein quantification in the SYKFT group after 48 weeks was 8.78% ± 2.56% (P = 0.006) more than that in the losartan 50 mg group, which was 0.51% ± 2.54% (P = 1.000) less than that in the losartan 100 mg group. Compared with the losartan potassium 50 mg group, the SYKFT plus losartan potassium 50 mg group had a 13.39% ± 2.49% (P < 0.001) greater reduction in urine protein level. Compared with the losartan potassium 100 mg group, the SYKFT plus losartan potassium 100 mg group had a 9.77% ± 2.52% (P = 0.001) greater reduction in urine protein. With a superiority threshold of 15%, neither was statistically significant. eGFR, serum creatinine and serum albumin from the baseline did not change statistically significant. The average change in TCM syndrome score between the patients who took SYKFT (-3.00 [-6.00, -2.00]) and who did not take SYKFT (-2.00 [-5.00, 0]) was statistically significant (P = 0.003). No obvious adverse reactions were observed in any group. CONCLUSION: SYKFT decreased the proteinuria and improved the TCM syndrome scores of primary glomerulonephritis patients, with no change in the rate of decrease in the eGFR. SYKFT plus losartan potassium therapy decreased proteinuria more than losartan potassium therapy alone. TRIAL REGISTRATION NUMBER NCT02063100 on ClinicalTrials.gov.

ObjectiveThe present study was conducted to investigate the protective effect on cardiac function and potential mechanism of Compound Danshen Dripping Pills （CDDPs） on myocardial ischemia reperfusion in rats. MethodsThirty two male SD rats were underwent cardiac reperfusion following 45 minutes of left anterior descending coronary artery ligation， and randomly divided into 4 groups （n= 8 ）， rats in each group were given different doses of CDDPs （40，80，120 mg·kg^-1·d^-1 ）， or normal saline （control group） by gavage. Another 8 rats underwent similar procedure but without LAD ligation were set as sham group （were also given same volume of normal saline by gavage）. The treatment lasted for 4 weeks. Then echocardiography was conducted to evaluate the end-point cardiac function. HE and Masson’s trichrome staining were performed to observe the change of histomorphology and fibrosis. CD31/α-SMA immunofluorescence was implemented to investigate the endothelial to mesenchymal transition in cardiac microvessels. Western Blot was used to analyze the expression of α-SMA and CD31 in ventricular tissue of infarcted border zone. Data were analyzed by one-way ANOVA or Kruskal-Wallis H non-parametric test. ResultsCompared with normal saline group， treatment with different doses of CDDP could increase ejection fraction and fractional shortening significantly （P＜0.05 at least）， decrease left ventricular end-diastolic volume and the E/A ratio significantly （P＜0.05 at least）， reduce the cardiac collagen volume fraction （both P<0.05）， and suppress the expression of mesenchymal marker α-SMA in cardiac microvessels detected by immunofluorescent staining（P＜0.05 at least）， and decrease the expression of α-SMA and increase the expression of CD31 in ventricular tissue of infarcted border zone detected by Western blot. ConclusionTreatment with 40/80/120 mg·kg^-1·d^-1 doses of CDDPs for 4 weeks could improve cardiac function in rats underwent ischemia-reperfusion， this might be through reducing the occurrence of endothelial to mesenchymal transition in microvessels within heart tissue， and subsequently decreasing the cardiac fibrosis.