Objective To optimize the technological conditions of polysaccharide purification from compound Qianyu water decoction. Methods Water extraction and alcohol precipitation, resolution, Sevag method and dialysis method were used to purify polysaccharide.The purity of polysaccharide was measured with the phenol-sulfuric acid spectrophotometry.On the basis of single factor test, effects of redissolved solid-liquid ratio, number of protein removal, and dialytic time on polysaccharide purity of Qianyu were investigated by orthogonal test. Results The best conditions for purification of polysaccharide in Qianyu were as follows: liquid-solid ratio was 1:40(g/mL, W/V), remove protein for 10 times, and dialysis for 18 h.The content of polysaccharide could reach 69.04%, and the transfer rate was 51.84%. Conclusion The optimized purification process was simple and accurate.It can be used for polysaccharide purification in compound Qianyu water decoction.

Objective:To evaluate the efficacy and safety of a China original liquid pulsation system for the treatment of meibomian gland dysfunction (MGD).Methods:A non-randomized controlled clinical trial was conducted.Twenty-two patients (44 eyes) diagnosed with MGD in Eye and ENT Hospital of Fudan University from February to August 2022 were enrolled.The patients were assigned into two groups according to their willingness.Of the 22 patients (44 eyes), 10 patients (20 eyes) in single liquid pulsation system group were treated with single liquid pulsation system for 12 minutes, and 12 patients (24 eyes) in intense pulsed light (IPL) group were treated with a course (4 times) of IPL, warm compresses and meibomian gland massage at three-week intervals.There was no difference in age and other baseline clinical indexes between the two groups (all at P<0.05). The meibum grading, quality grading of tear film lipid layer, Symptom Assessment Questionnaire in Dry Eye (SANDE) questionnaire score, first and average tear breakup time (BUT), corneal fluorescein sodium staining (CFS) score, tear meniscus height (TMH), and the area of meibomian gland loss were determined at baseline, 1 and 3 months after treatment.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Eye and ENT Hospital of Fudan University (No.2021069). Written informed consent was obtained from each patient before any medical examination. Results:Statistically significant group effects and time effects were found in the quality of tear film lipid layer ( Hgroup=4.39, P=0.036, Htime=6.30, P=0.043) and average BUT ( Fgroup=4.41, P=0.038; Ftime=4.08, P=0.049) in the two groups.The meibum grading, first BUT and TMH 1 and 3 months after treatment were significantly better than before treatment in single liquid pulsation system group (all at P<0.05). Compared with before treatment, there was no significant improvement in the meibum grading, distribution of tear film lipid, first BUT and TMH at 1 and 3 months after treatment in IPL group (all at P>0.05). In both groups, the SANDE and CFS scores 1 and 3 months after treatment were better than those before treatment, showing statistically significance (all at P<0.05). In terms of safety, neither instrument-related adverse events nor extra complaints of discomfort were reported in the single liquid pulsation system group.In both groups, the number of patients with positive CFS staining significantly decreased, and no new cases with positive CFS appeared after treatment. Conclusions:This China original liquid pulsation system is a safe and effective physical therapy in improving tear film dysfunction and ocular surface symptoms of MGD patients within 3 months after treatment.

Objective:To investigate the high-risk factors affecting the prognosis of patients with pT 1-2N 1M 0 after mastectomy, establish a nomogram prediction model, perform risk stratification, and screen the radiotherapy benefit populations. Methods:Clinical data of 936 patients with pT 1-2N 1M 0 breast cancer undergoing mastectomy in the Fourth Hospital of Hebei Medical University from January 2010 to December 2016 were retrospectively analyzed and 908 cases had complete follow-up data. They were divided into the radiotherapy (RT) group ( n=583) and non radiotherapy (NRT) group ( n=325) according to the radiotherapy. After propensity score matching (PSM) was performed 1 vs. 1, 298 cases were assigned into the RT group and 298 in the NRT group. Overall survival (OS) and disease-free survival (DFS) were compared between two groups using log-rank test. Nomogram prediction model was established, the survival differences were compared among different risk groups, and the radiotherapy benefit populations were screened. Results:Univariate analysis showed that the 5- and 8-year OS and DFS in the RT group were significantly better than those in the NRT group (both P<0.001). Multivariate analysis showed that age, tumor quadrant, number of lymph node metastases, T staging, and Ki-67 level were the independent prognostic factors for OS. Age, tumor quadrant, and T staging were the independent prognostic factors for DFS. The OS nomogram analysis showed that the OS of patients in the high-risk group was significantly improved by post-mastectomy radiotherapy (PMRT) ( P=0.001), while PMRT did not show an advantage in the low- and medium-risk groups ( P=0.057, P=0.099). The DFS nomogram analysis showed that DFS was significantly improved by PMRT in patients in the medium- and high-risk groups ( P=0.036, P=0.001), whereas the benefits from PMRT were not significant in the low-risk group ( P=0.475). Conclusions:For patients with pT 1-2N 1M 0 breast cancer after mastectomy, age ≤ 40 years, tumor located in the inner quadrant or central area, T 2 staging, 2-3 lymph node metastases, Ki-67>30% are the high-risk factors affecting clinical prognosis. The nomogram prediction model can screen the populations that can benefit from PMRT, providing reference for clinical decision-making.

OBJECTIVE To explore the difference in th e mechanis m of baicalein and wogonin inhibiting the energy metabolism of hepatoma cells. METHODS Human hepatoma HepG 2 cells were divided into blank control group （without medicine），different dose groups of baicalein and wogonin （1.25，2.5，5，10 and 20 μmol/L）. The effects of baicalein and wogonin on the viability of HepG 2 cells were detected by MTT assay. HepG 2 cells were divided into blank control group （without medicine），baicalein group and wogonin group. After administration ，the concentration of ATP in cell was detected by enhanced ATP kit. The levels of cell glycolysis and mitochondrial energy metabolism were evaluated by glycolysis and mitochondrial pressure test kit ；the affinity of baicalein and wogonin with key enzymes of energy metabolism was predicted by molecular docking ，and the key enzymes of energy metabolism with high affinity were screened ；the expression of key enzymes of energy metabolism was detected by Western blot. RESULTS Within the dose range of 2.5-20 μmol/L，the half inhibitory concentrations of baicalein and wogonin were 12.84 and 24.09 μmol/L；baicalein 1.25 μmol/L and wogonin 2.5 μmol/L had no effect on cell viability ，so it was selected as the dosage for subsequent experiments. Compared with blank control group ，the concentration of ATP in HepG 2 cells decreased significantly in baicalein group and wogonin group （P＜0.05）；the inhibitory effects on basic acidification rate of HepG 2 cells in wogonin group were significantly stronger than those of baicalein group （P＜0.05），but there was no significant difference between them on the basic oxygen consumption rate （P＞0.05）；baicalein had strong binding to pyruvate kinase M 2 and mitochondrial enzyme complexes Ⅰ（CⅠ），C Ⅱ and C Ⅳ，while wogonin only had strong binding to pyruvate kinase M 2； wogonin could significantly down-regulate the protein expressions of hexokinase ，phosphofructokinase，pyruvate kinase M 2，CⅠ， C Ⅱ and C Ⅳ（P＜0.05），but there was no statistical significance in the effect of baicalein on the regulation of these enzymes （P＞ 0.05）. CONCLUSIONS Both baicalein and wogonin can inhibit the energy metabolism of hepatoma HepG 2 cells，but the mechanism is different ：the effect of baicalein is related to the activity of key enzymes ，while the effect of wogonin is related to the inhibition of the expression of key enzymes of energy metabolism.

OBJECTIVE：To establish the method for the content de termination of 17 quality markers in Dahuang zhechong pills（DHZCP）. METHODS ：HPLC method was adopted to determine the contents of 17 quality markers in 10 batches of DHZCP ， such as allantoin ，hypoxanthine，salidroside，hydroxypaeoniflorin，glycyrrhizin，isoglycyrrhizin，baicalin，p-methoxyphenylacetic acid，wogonin，cinnamic acid ，apigenin，naringin，norwogonin，aloe emodin ，rhein，chrysin，emodin. The determination was performed on Kromasil 100-5-C18（250 mm × 4.6 mm，5 μm）column with mobile phase consisted of 0.1％ phosphoric acid solution-acetonitrile （gradient elution ） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃ ，the detection wavelength was 210 nm and the sample size was 20 μ L. RESULTS：The linear range of above 17 quality markers were 5.74-183.53，6.51-208.24，4.30-137.65，4.60-147.06，4.12-131.76，4.25-135.88，6.31-201.76，4.60-147.06，1.94-62.06，4.47- 142.94，0.69-22.06，2.29-73.24，2.33-74.41，1.42-45.29，6.65-212.94，1.11-35.44 and 1.47-47.06 μg/mL，respectively（all R2≥ 0.999 0）. RSDs of precision ，repeatability，stability and durability tests were all less than 2％（n＝6）；average recovery of 17 quality markers ranged from 96.31％ to 101.73％，and the RSDs were less than 3％（n＝6）. CONCLUSIONS ：The method is simple， rapid，speific，specise，reproducible，stable，accurate and durable ，and can be used for improving the quality standard of DHZCP.

BACKGROUND: Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. METHODS: The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. RESULTS: Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. CONCLUSIONS This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/radiotherapy* ; Cell Cycle Proteins/metabolism* ; Protein-Tyrosine Kinases/genetics* ; Apoptosis ; Programmed Cell Death 1 Receptor ; Cell Line, Tumor ; G2 Phase Cell Cycle Checkpoints ; Liver Neoplasms/radiotherapy* ; Tumor Microenvironment ; Pyrazoles ; Pyrimidinones

ObjectiveTo investigate the influence of different diagnostic criteria on the short-term prognosis of patients with acute-on-chronic liver failure （ACLF）. MethodsA total of 115 ACLF patients who were hospitalized in Department of Gastroenterology， The Second Affiliated Hospital of Kunming Medical University， from January 2018 to January 2022 were enrolled， and all patients received internal medical treatment combined with artificial liver therapy. According to the guidelines， the patients were divided into CMA guideline group （Diagnostic and treatment guidelines for liver failure by Chinese Medical Association）（n=100）， APASL guideline group （Consensus statements of Asian Pacific Association for the Study of the Liver）（n=94）， and EASL guideline group （Criteria proposed by European Association for the Study of the Liver）（n=36）. The above three guidelines were compared in terms of 90-day mortality rate. A one-way analysis of variance was used for comprision of continuous date between groups； the chi-square test was used for comprision of categorical date between groups. The receiver operating characteristic （ROC） curve of related variables. ResultsThe 90-day mortality rate was 50.0% in the CMA guideline group， 51.1% in the APASL guideline group， and 77.8% in the EASL guideline group， and the EASL guideline group had a significantly higher 90-day mortality rate than the CMA guideline group （χ²=8.351， P=0.004） and the APASL guideline group （χ²=7.650， P=0.006）. EASL guideline had a sensitivity of 22.2% and a specificity of 92.3% in predicting the risk of short-term mortality， with an area under the ROC curve was 0.576. ConclusionACLF patients who meet EASL guideline tend to have a worse short-term prognosis， and this guideline may help to identify patients at a relatively high risk of short-term death.

[摘 要] 目的：探讨在靶向HER2的CAR的CD3ζ链胞内区引入YRHQ基序对CAR-T细胞的特异性杀伤活性及免疫记忆形成的影响。方法：通过DNA合成获得包含靶向HER2的编码抗原受体H28ζ或H28ζ(YRHQ)的DNA片段，通过慢病毒载体将不同CAR的DNA片段分别转导健康人外周血T细胞，制备靶向HER2的H28ζ-CAR-T及H28ζ(YRHQ)-CAR-T细胞。扩增过程中对不同CAR-T细胞进行计数，FCM检测CAR的表达率。将CAR-T细胞分别与HER2阳性的SKOV3、MDA-MB-453或HER2阴性的MCF-7细胞共培养，LDH释放法检测其杀伤活性，ELISA法检测IL-2、IFN-γ和颗粒酶B的水平，WB法检测STAT3磷酸化水平及免疫检查点分子TIM-3和PD-1的表达，通过FCM检测CCR7、CD45RO的表达，分析CAR-T细胞的表型。结果：H28ζ-CAR-T和H28ζ(YRHQ)-CAR-T细胞扩增能力较好，体外培养7 d时扩增4~5倍。H28ζ-CAR和H28ζ(YRHQ)-CAR表达率分别为（33.3±2.85）%和（28.30±3.2）%。H28ξ(YRHQ)-CAR-T细胞的杀伤活性较H28ζ-CAR-T细胞更高（P<0.05）。经HER2抗原刺激后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞的STAT3磷酸化水平较H28ξ-CAR-T细胞明显升高（P<0.01）；而两者间PD-1和TIM-3的表达无明显差异。未经抗原刺激的CAR-T细胞CCR7和CD45RO表达与正常T细胞比较差异无统计学意义（均P>0.05），与SKOV3细胞共培养后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞中TEM细胞比例明显增加、TCM细胞比例明显减少（均P<0.05）。结论：在CD3胞内区引入YRHQ基序可在一定程度上提高CAR-T细胞的杀伤潜力。