ObjectiveTo investigate the mechanism of modified Si Junzitang and Shashen Maidong Tang ［Yiqi Yangyin Jiedu prescription （YQYYJD）］ in enhancing the sensitivity of cisplatin in epidermal growth factor receptor tyrosine kinase inhibitor （EGFR-TKI）-resistant lung adenocarcinoma cells based on aerobic glycolysis. MethodsThe effects of different concentrations of YQYYJD （0， 2， 3， 4， 5， 6， 7， 8 g·L^-1） and cisplatin （0， 3， 6， 9， 12， 15， 18， 21， 24， 27 mg·L^-1） on the proliferation and activity of PC9/GR cells were detected by the cell counting kit-8 （CCK-8） assay after 24 hours of intervention. The half-maximal inhibitory concentration （IC₅₀） for PC9/GR cells was calculated to determine the concentrations used in subsequent experiments. PC9/GR cells were divided into blank group （complete medium）， YQYYJD group （5 g·L^-1）， cisplatin group （12 mg·L^-1）， and combined group （YQYYJD 5 g·L^-1 + cisplatin 12 mg·L^-1）. After 24 hours of intervention， cell viability was measured using CCK-8 assay. Cell proliferation was assessed by colony formation assay， and cell migration was evaluated by scratch and Transwell assays. Glucose consumption， lactate production， and adenosine triphosphate （ATP） levels were measured by colorimetric assays. The expression levels of glycolysis-related proteins， including hexokinase 2 （HK2）， phosphofructokinase P （PFKP）， pyruvate kinase M2 （PKM2）， lactate dehydrogenase A （LDHA）， glucose transporter 1 （GLUT1）， and monocarboxylate transporter 4 （MCT4）， were determined by Western blot. ResultsBoth YQYYJD and cisplatin inhibited the viability of PC9/GR cells in a concentration-dependent manner. The IC₅₀ of PC9/GR cells for YQYYJD and cisplatin were 5.15 g·L^-1 and 12.91 mg·L^-1， respectively. In terms of cell proliferation， compared with the blank group， the cell survival rate and the number of colonies formed in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group showed a further significant reduction in cell survival rate and colony formation （P<0.01）. In terms of cell migration， compared with the blank group， the cell migration rate and the number of cells passing through the Transwell membrane in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group exhibited a further significant reduction in cell migration rate and the number of cells passing through the Transwell membrane （P<0.01）. In terms of glycolysis， compared with the blank group， glucose consumption， lactate production， and ATP levels in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group showed a further significant reduction in glucose consumption， lactate production， and ATP levels （P<0.05）. Compared with the blank group， the protein expression levels of HK2， PFKP， PKM2， and LDHA in the YQYYJD， cisplatin， and combined groups were significantly decreased （P<0.01）. The combined group showed a further significant reduction in the expression levels of these proteins compared with the YQYYJD and cisplatin groups （P<0.01）. No significant differences were observed in the protein expression levels of GLUT1 and MCT4 among the groups. ConclusionYQYYJD can synergistically inhibit the proliferation and migration of PC9/GR cells and enhance their sensitivity to cisplatin. The mechanism may be related to the downregulation of the expression of glycolysis-related rate-limiting enzymes， including HK2， PFKP， PKM2， and LDHA， thereby inhibiting glycolysis.

ObjectiveTo observe the prognosis effect of soluble programmed death ligand-1（sPD-L1） in treating patients with advanced lung adenocarcinoma treated with epidermal growth factor receptor-tyrosine kinase inhibitors（EGFR-TKIs） and the influence of Yiqi Yangyin Jiedu prescription. MethodA prospective cohort-controlled study was conducted to enroll patients treated with EGFR-TKIs in the first line of treatment，who were admitted to the Oncology Department of Longhua Hospital and Shanghai Chest Hospital from May 1^st， 2021 to June 30^th， 2023， and they were evaluated as non-progressive and identified with deficiency of Qi and Yin after one month of treatment. The patients were divided into an exposed group （EGFR-TKIs combined with Yiqi Yangyin Jiedu prescription） and a non-exposed group （EGFR-TKIs alone）according to whether or not they were treated with Yiqi Yangyin Jiedu prescription and were treated until disease progression， or death and intolerable adverse reactions occurred. The enzyme-linked immunosorbent assay （ELISA） was applied to detect the level of sPD-L1 in patients at the time of enrollment and disease progression，and Cox risk proportionality model was used to analyze the independent prognostic factors affecting disease progression of patients treated with EGFR-TKIs. ResultA total of 90 patients （39 in the exposed group and 51 in the non-exposed group） undergoing disease progression after EGFR-TKI treatment were enrolled. At the time of enrolment and after disease progression，the levels of serum sPD-L1 in the 90 patients were 12.06 （27.54） ng·L^-1 and 41.99 （62.93） ng·L^-1，respectively. Compared with that at the time of enrollment， the serum sPD-L1 level in the 90 patients was significantly increased after disease progression （P<0.01）. The serum sPD-L1 level in patients in the exposed group was 12.27 （24.78） ng·L^-1 and 29.57 （61.12）ng·L^-1 respectively at the time of enrolment and after disease progression. In the non-exposed group， patients had serum sPD-L1 levels of 11.81 （28.46） ng·L^-1 and 49.54 （74.12） ng·L^-1 respectively at the time of enrolment and after disease progression. Compared with that at the time of enrollment， the serum sPD-L1 level in the two groups of patients was significantly increased after disease progression （P<0.01）. In addition， compared with that in the non-exposed group， the sPD-L1 level in the exposed group was greatly reduced after disease progression（P<0.01）. Cox multifactorial analysis showed that sPD-L1 level and age at the time of enrolment were associated with patients' progression-free survival（PFS），and that low levels of sPD-L1 （<12.06 ng·L^-1） prolonged the PFS and reduced the risk of disease progression in patients treated with EGFR-TKIs compared with high levels of sPD-L1. ConclusionElevated sPD-L1 level is a poor prognostic factor for the long-term efficacy of EGFR-TKIs，and treatment with Yiqi Yangiin Jiedu prescription can down-regulate sPD-L1 level of patients treated with EGFR-TKIs.

[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

OBJECTIVES: To compare the efficacy of toric implantable collamer lens (Toric-ICL) and femtosecond laser-assisted in situ keratomileusis (FS-LASIK) for myopia correction in patients with moderate to high myopia complicated with astigmatism. METHODS: We retrospectively collected data from 64 patients (aged 18-42 years) with moderate to high myopia complicated with astigmatism (128 eyes) undergoing either Toric-ICL (28 patients/56 eyes) or FS-LASIK (36 patients/72 eyes) at our department between January, 2019 and December, 2020. The changes of uncorrected distance visual acuity (UCVA), spherical equivalent (SE), mean astigmatism correction index (CI), corneal endothelial cell density (ECD) and intraocular pressure (IOP) following the procedures were compared between the two groups. RESULTS: In FS-LASIK group, all the eyes (72/72) achieved an UCVA≥1.0, similar to the rate in Toric-ICL group (55/56 eyes; P=0.2374). The postoperative SE was also comparable between FS-LASIK and Toric-ICL groups [0.43±0.06 D (range: -1.0 to 1.50 D) vs 0.38±0.05 D (range: -0.75 to 1.00 D); P=0.56]. The mean astigmatism CI was significantly higher in FS-LASIK group than in Toric-ICL group (0.8561 vs 0.7176; P<0.0001), and 88.89% of the eyes in FS-LASIK group and 69.64% in Toric-ICL group had postoperative astigmatism ≤0.50 D. No significant changes were observed in postoperative corneal ECD in FS-LASIK group, whereas ECD decreased significantly after the procedure in Toric-ICL group (P=0.0057). The patients undergoing Toric-ICL exhibited no significant changes of postoperative IOP, but the patients receiving FS-LASIK had significantly reduced IOP after the procedure (P<0.001). CONCLUSIONS Although the patients included in Toric-ICL group had higher myopia and astigmatism, Toric-ICL still showed better predictability and efficacy for astigmatic correction in Toric-ICL group. Toric-ICL is an effective and safe equivalent of FS-LASIK for correcting moderate myopia but can be more advantageous for correcting high myopia with astigmatism.
Humans ; Astigmatism/complications* ; Myopia/complications* ; Keratomileusis, Laser In Situ/methods* ; Retrospective Studies ; Adult ; Visual Acuity ; Adolescent ; Young Adult ; Treatment Outcome ; Male ; Lens Implantation, Intraocular/methods* ; Female ; Phakic Intraocular Lenses ; Intraocular Pressure

Objective: To analyze the frequency and investigate the causes of unexpected antibodies in D-negative blood donors. Methods: From January 2022 to December 2024, 3 768 D-negative blood donors sent to our laboratory were selected as research subjects. D-negative confirmation test and RhCE phenotype detection were applied by saline tube method and microcolumn gel indirect antiglobulin test (IAT), respectively. Antibody screening and identification were performed using the polybrene method and IAT column agglutination methods. Anti-D, anti-C and anti-G specificities were identified by a two-step adsorption-elution method, and the genotypes of D-negative samples were determined by RHD gene amplification, Sanger sequencing, and PacBio Single Molecule Real-Time (SMRT) sequencing. Results: Among D-negative donors, ccee and Ccee phenotypes accounted for the highest proportion, 55.68% (2 098/3 768) and 29.56% (1 114/3 768), respectively, while CcEE and CCEe phenotypes were the least, with one case detected in each, accounting for 0.03% (1/3 768). A total of 165 cases with D variant phenotype were detected, and the proportion of D variant was 4.38% (165/3 768) in the donors detected by D-negative confirmation test. Antibody screening positive blood donors were identified in 93 cases with a proportion of 2.47% (93/3 768). Antibody specificity was determined in 84 blood donors, and 9 samples showed no clear specificity. Anti-D was detected most frequently (n=68), in which 6 of them were detected having multiple antibodies, anti-D + anti-C (n=2), anti-D + anti-G(n=1), and anti-D + anti-E(n=3). The other antibodies detected were anti-E (n=1), anti-M(n=9), anti-P1 (n=3), anti-Le (n=1), and anti-HI(n=2). Fourteen cases were detected with anti-D in serological D-negative donors with C+ or E+ phenotype, in which three of them were DVI type 3 individuals and 11 cases were D negative individuals. Conclusion: The incidence of unexpected antibodies was higher in D-negative blood donors than in the total donors, with anti-D being the most common. The data provide insights for prevention and monitoring hemolytic disease of the fetus and newborn (HDFN) caused by anti-D. To ensure the safety of blood transfusion, routine unexpected antibody screening for RhD-negative blood donors is recommended to prevent the use of unexpected antibodies positive plasma in the clinic.

ObjectiveTo investigate the effect and mechanism of ginsenoside Rg₁ （G-Rg₁） combined with hirudin in treating myocardial fibrosis in the mouse model of acute myocardial infarction （AMI）. MethodSeventy-five C57BL/6N mice were randomized into sham， model， G-Rg₁ （20 mg·kg^-1）， hirudin （20 mg·kg^-1）， and G-Rg₁ （20 mg·kg^-1） + hirudin （20 mg·kg^-1） groups. The mouse model of AMI was established by ligation of the anterior descending branch of the left coronary artery and continued gavage for 4 weeks. The success of the modeling was judged by ECG changes of mice before and after ligation. The heart weight index， echocardiography， myocardial fibrosis， type Ⅰ collagen α1（COL1A1）， platelet-endothelial cell adhesion molecule-1 （PECAM-1/CD31）， α-smooth muscle actin （α-SMA）， intercellular adhesion molecule-1 （ICAM-1）， and vascular cell adhesion molecule-1 （VCAM-1） were measured before and after treatment. ResultAfter ligation of the anterior descending branch of the left coronary artery， the R wave and T wave merged into a tall tented wave， and the ST segment presented a "damaged" change， indicating that the model was successfully prepared. Compared with the sham group， the model group showed dull and dry hair， slow movement， increased heart weight index （P<0.01）， decreased left ventricular ejection fraction （EF） and left ventricular shortening fraction （FS） （P<0.01）， increased left ventricular end-diastolic diameter （LVEDd） and left ventricular end-systolic diameter （LVESd） （P<0.01）， disarranged myocardial fibers， collagen fiber hyperplasia （P<0.01）， increased expression of COL1A1 （P<0.01）， upregulated protein levels of ICAM-1 and VCAM-1 （P<0.01）， downregulated CD31 expression， and upregulated α-SMA expression （P<0.01）. Compared with the model group， the treatment groups recovered the above indexes in different degrees （P<0.05， P<0.01）， and the combined group had better effect （P<0.05）. ConclusionG-Rg₁ combined with hirudin can ameliorate myocardial fibrosis after AMI by inhibiting the expression of adhesion molecular protein in the heart tissue， reducing the adhesion of inflammatory cells， alleviating cardiac inflammation， and inhibiting cardiac endothelial-to-mesenchymal transition.

Objective To evaluate the molluscicidal effect of spraying different formulations of niclosamide ethanolamine salt with drones against Oncomelania hupensis in ditches. Methods A semi-dry and semi-wet ditch with O. hupensis snails was selected in the second branch field of Jiangbei Farm, Jiangling County, Hubei Province in May 2023, and divided into 4 experimental areas, named groups A1, A2, B1 and B2. Environmental cleaning was performed in groups A1 and B2, and was not conducted in groups A2 or B2. Then, 50% wettable powder of niclosamide ethanolamine salt was sprayed with drones at an effective dose of 2 g/m² in groups A1 and A2, and 5% niclosamide ethanolamine salt granule was sprayed with drones at an effective dose of 2 g/m² in groups B1 and B2. O. hupensis snails were surveyed using the systematic sampling method 1, 3, 5, 7, 14 days after spraying, and the natural mortality and corrected mortality of O. hupensis snails were calculated. Results The occurrence of frames with living snails, mean density of living snails and natural mortality of snails were 97.50% (117/120), 6.30 snails/0.1 m² and 1.18% (9/765) in the test ditch before spraying, respectively. There were significant differences in the mortality of snails among four groups 1, 3, 5, 7 and 14 days after spraying niclosamide formulations with drones (χ² = 17.230, 51.707, 65.184, 204.050 and 34.435, all P values < 0.01). The overall mortality rates of snails were 94.51% (1 051/1 112), 79.44% (908/1 143), 96.54% (977/1 012) and 88.55% (1 021/1 153) in groups A1, A2, B1 and B2 (χ² = 207.773, P < 0.05), respectively. In addition, there was no significant difference in the overall snail mortality between groups A1 and B1 (P > 0.05), and the snail mortality in groups A1 and B1 were both statistically different from that in groups A2 and B2 (all P values < 0.05). Conclusion Both 50% wettlable powder of niclosamide ethanolamine salt and 5% niclosamide ethanolamine salt granule sprayed with drones are active against O. hupensis snails in ditches, and environmental cleaning may improve the molluscicidal effect.

OBJECTIVE To study the chemical composition and the function of solid excipients in relieving the "nourishing and spleen-impairing" effect of Zhangbang nine steaming nine sun-dying Rehmanniae Radix(RR) by using UPLC-Q-TOF-MS technique. METHODS UPLC-Q-TOF-MS was used to collect the data of Zhangbang nine steaming nine sun-dying RR, and UNIFI platform and self-built database were used for the rapid qualitative analysis and identification of the chemical components of Zhangbang nine steaming nine sun-dying RR. The effect of solid excipients on the function of RR "nourishing and spleen-impairing" was investigated by establishing a mouse model of low gastric motility and measuring its gastric residual rate and small intestine propulsion rate. RESULTS The UPLC-Q-TOF-MS and UNFI platforms identified 76 chemical components in nine steaming nine sun-dying RR, including 9 iridoid glycosides, 11 phenylethyl glycosides, 31 flavonoids, 6 saccharides and 19 other compounds; the results of gastric emptying and small intestinal propulsion experiments on mice showed that Zhangbang nine steaming nine sun-dying RR had gastric emptying and intestinal propulsion effects. CONCLUSION This method can be used for the rapid identification and analysis of nine steaming nine sun-dying RR, which has solid excipients Citri Reticulatae Pericarpium and Amomi Fructus to alleviate the "nourishing and spleen-impairing" function of RR. This study can provide a reference for quality control and elucidation of the material basis of the medicinal effects of specialty beverage of Zhangbang nine steaming nine sun-dying RR.

Objective: To investigate the changes of microorganisms and metabolites in joint effusion of patients with Rheumatoid arthritis(RA), Osteoarthritis(OA) and Urarthritis(UA). To provide new ideas for the study of the effect of microbiota on the pathogenesis of arthritis. Methods: Joint effusion samples were collected from 20 patients with RA, 20 patients with OA, and 20 patients with UA. 16S rRNA gene sequencing and untargeted ultra-high performance Liquid chromatography-mass spectrometry(LC-MS) were used to explore the differences in microorganisms and metabolites among the three groups. Pearson correlation analysis was used to detect the correlation between effusion microbiota and metabolites. Results: There were differences in microbial diversity and microbiota composition among the three groups. Combined with VIP>1 from OPLS-DA andP<0.05 from two-tailed Students t-test, 45 differential metabolites(Between RA and OA groups), 38 differential metabolites(Between UA and OA groups) and 16 differential metabolites(Between RA and UA groups), were identified. GO analysis and KEGG pathway analysis showed that the differential metabolic pathways among the three groups were mainly concentrated in citric acid cycle(TCA cycle), nucleotide metabolism, amino acid metabolism and glycolysis pathway. Correlation analysis of joint effusion microbiota and metabolites suggested that bacteria enriched in the three groups of joint effusion, such asPrevotella,Clostridium ruminosus,Prevotellaceae＿UCG-001, were related to many key metabolites such as lysozyme, uric acid, glucose, and L-glutamine. Conclusion This study shows that there are a variety of bacterial flora in joint cavity effusion of RA, OA, and UA patients, and the differential metabolites produced by them are involved in the pathogenesis of the three types of arthritis by affecting a variety of metabolic pathways.