Objective:To develop a convolutional neural network model of whole slide imaging of cerebrospinal fluid cells for rapid and accurate identification and classification of tumor cells in cerebrospinal fluid.Methods:A total of 8 692 cerebrospinal fluid cytology smears from Huashan Hospital Affiliated to Fudan University from January 2nd, 2019, to December 27th, 2024. As randomly assigned, the training set included 4 941 benign and 1 745 malignant samples, while the validation set comprised of 1 368 benign and 638 malignant samples. Whole-slide digital images were acquired using a cytopathology scanner, cells (clusters) were annotated for classification, and a deep learning model was constructed via tiled image patches for cell detection and classification. Model performance was evaluated using accuracy, sensitivity, specificity, and other indicators. The classification efficiency of manual microscopy was compared.Results:The model achieved a mean precision of 96.75% for cerebrospinal fluid cell classification. For malignant tumor cells, the classification accuracy was 96.61% (mAP=98.36%, AUC=0.97). Subtype classification accuracies for epithelial/epithelioid tumors and small round cell tumors were 97.13% (AUC=0.98) and 95.58% (AUC=0.93), respectively. Compared with manual microscopy, which took (9.70±0.82) minutes for classifying 200 cells, (18.27±1.21) minutes for 500 cells, and often exceeded 60 minutes or infeasible for full slides, the AI model took (3.46±0.49) seconds for 200 cells, (6.76±0.82) seconds for 500 cells, and a median of 48.57 seconds for full slides ( P<0.001), representing an efficiency improvement of approximately 161-170 times, significantly enhancing diagnostic efficiency. Conclusion:This fully automated hierarchical deep learning model enables efficient and accurate tumor cell identification and classification in CSF, providing an effective auxiliary tool for the rapid diagnosis of meningeal carcinomatosis.

Objective:To develop a convolutional neural network model of whole slide imaging of cerebrospinal fluid cells for rapid and accurate identification and classification of tumor cells in cerebrospinal fluid.Methods:A total of 8 692 cerebrospinal fluid cytology smears from Huashan Hospital Affiliated to Fudan University from January 2nd, 2019, to December 27th, 2024. As randomly assigned, the training set included 4 941 benign and 1 745 malignant samples, while the validation set comprised of 1 368 benign and 638 malignant samples. Whole-slide digital images were acquired using a cytopathology scanner, cells (clusters) were annotated for classification, and a deep learning model was constructed via tiled image patches for cell detection and classification. Model performance was evaluated using accuracy, sensitivity, specificity, and other indicators. The classification efficiency of manual microscopy was compared.Results:The model achieved a mean precision of 96.75% for cerebrospinal fluid cell classification. For malignant tumor cells, the classification accuracy was 96.61% (mAP=98.36%, AUC=0.97). Subtype classification accuracies for epithelial/epithelioid tumors and small round cell tumors were 97.13% (AUC=0.98) and 95.58% (AUC=0.93), respectively. Compared with manual microscopy, which took (9.70±0.82) minutes for classifying 200 cells, (18.27±1.21) minutes for 500 cells, and often exceeded 60 minutes or infeasible for full slides, the AI model took (3.46±0.49) seconds for 200 cells, (6.76±0.82) seconds for 500 cells, and a median of 48.57 seconds for full slides ( P<0.001), representing an efficiency improvement of approximately 161-170 times, significantly enhancing diagnostic efficiency. Conclusion:This fully automated hierarchical deep learning model enables efficient and accurate tumor cell identification and classification in CSF, providing an effective auxiliary tool for the rapid diagnosis of meningeal carcinomatosis.

Objective To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection, and to examine the effect of oxymatrine (OMT) on C. parvum infection in mice. Methods Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 10⁵ C. parvum oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model C. parvum intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of HMGB1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay. Results HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height (F = 6.207, P = 0.000 5), intestinal crypt depth (F = 6.903, P = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth (F = 37.190, P < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) μm vs. (399.5 ± 30.9) μm; t = 4.178, P < 0.01] and the GA group [(321.9 ± 41.1) μm vs. (383.7 ± 42.7) μm; t = 3.130, P < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) μm] than in the control group [(128.4 ± 23.6) μm] (t = 3.877, P < 0.01) and GA group [(143.3 ± 24.7) μm] (t = 2.710, P < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) μm] than in the infection group (t = 3.888, P < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t = 1.989, P > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) μm] than in the infection group (t = 4.133, P < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t = 0.575, P > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) (t = 10.540, P < 0.01) and the GA group (2.7 ± 0.3) (t = 7.370, P < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group (t = 15.020, P < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t = 0.404, P > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F = 28.031, P < 0.000 1) and ZO1 expression (F = 14.122, P < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; t = 3.810, P < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group (t = 7.620 and 5.391, both P values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 1.791 and 2.033, both P values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; t = 4.485, P < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] (t = 5.159, P < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] (t = 4.441, P < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 0.037 and 0.742, both P values > 0.05). qPCR assay showed significant differences among the four groups in terms of HMGB1 (F = 21.980, P < 0.000 1), TLR2 (F = 20.630, P < 0.000 1), TLR4 (F = 17.000, P = 0.000 6), MyD88 (F = 8.907, P = 0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F = 8.889, P = 0.000 7). The relative expression of HMGB1 [(5.97 ± 1.07) vs. (1.05 ± 0.07); t = 6.482, P < 0.05] 、TLR2 [(5.92 ± 1.29) vs. (1.10 ± 0.14); t = 5.272, P < 0.05] 、TLR4 [(5.96 ± 1.50) vs. (1.02 ± 0.03); t = 4.644, P < 0.05] 、MyD88 [(3.00 ± 1.26) vs. (1.02 ± 0.05); t = 2.734, P < 0.05] and NF-κB p65 mRNA [(2.33 ± 0.72) vs. (1.04 ± 0.06); t = 2.665, P < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of HMGB1 (0.63 ± 0.01), TLR2 (0.42 ± 0.10), TLR4 (0.35 ± 0.07), MyD88 (0.70 ± 0.11) and NF-κB p65 mRNA (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group (t = 8.629, 5.830, 11.500, 4.729 and 6.898, all P values < 0.05), and the relative expression of HMGB1, TLR2, TLR4, MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t = 7.052, 6.035, 4.084, 3.165 and 3.274, all P values < 0.05). In addition, the relative expression of HMGB1 (1.14 ± 0.60), TLR2 (1.00 ± 0.24), TLR4 (1.14 ± 0.07), MyD88 (0.96 ± 0.25) and NF-κ B p65 mRNA (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t = 7.059, 5.320, 3.510, 3.466 and 3.273, all P values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1, TLR2, TLR4, MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t = 0.239, 0.518, 1.887, 0.427 and 0.641, all P values > 0.05). Conclusions C. parvum infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.

Objective:To investigate the clinical efficacy of 125I seed implantation combined with anlotinib hydrochloride in the treatment of non-small cell lung cancer (NSCLC). Methods:61 cases of NSCLC patients were enrolled, of which 30 cases (observation group) received 125I seed implantation combined with anlotinib treatment, and 31 cases (control group) received 125I seed implantation only. To evaluate the curative effect and adverse reactions of all patients, the carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), neuroendocrine enolase (NSE), squamous cell carcinoma antigen (SCC) in the peripheral blood of the two groups was measured before the treatment and at 1 and 3 months after the treatment. Results:The effective rates in the observation group were 90.00% and 93.33%, the effective rates in the control group were 67.74% and 74.19% at 1 and 3 months after the treatment, respectively, and the difference in efficacy between the two groups was statistically significant ( χ2=4.504, P=0.034 vs. χ2=4.075, P=0.044). There was no significant difference in the incidence of adverse reactions between the two groups of patients after treatment ( P=0.785). At 1 and 3 months after the treatment, the levels of CEA, CYFRA21-1, NSE and SCC in the peripheral blood of the two groups of patients were lower than those before the treatment (all P<0.05). Conclusions:125I seed implantation combined with anlotinib hydrochloride is safe for the treatment of advanced non-small cell lung cancer, and has promotion value.

【Objective】 To explore the protective effects of exogenous hydrogen sulfide （H2S） on oxygen glucose deprivation and re-oxygenation （OGD/R）-induced SH-SY5Y cell injury. 【Methods】 OGD/R model was established in SH-SY5Y cells. Based on interferences， SH-SY5Y cells were divided into control group （no interference）， OGD/R model group， and treatment groups （OGD/R model treated with different concentrations of NaHS （H2S donor）. The cells were cultured in low glucose medium without fetal bovine serum under 37 ℃， 93% N2 and hypoxia （2% O2） for 12 h， then in complete medium under normoxia （5% O2 ） for 24 h. CCK-8 was used to detect cell viability， and flow cytometry was used for assaying the level of cellular apoptosis. Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins including glucose regulatory protein 78 （GRP78）， CCAAT/enhancer-binding homologous protein （CHOP）， NF-κB P65 and COX-2. 【Results】 The cell viability was lower in OGD/R model group than in the control group （P<0.05）. NaHS treatment at a concentration of 0-0.5 mmol/L increased cell viability in OGD/R model group in a dose-dependent manner （P<0.05）. In contrast， NaHS （C>0.5 mmol/L） decreased cell viability of OGD/R （P<0.05）. The apoptosis level of early stage in OGD/R model group was significantly higher than that in control group （P<0.05）. NaHS treatment at concentrations of 0.2 and 0.4 mmol/L lowered the apoptosis level of early stage in OGD/R model group （P<0.05）. Western blot showed that the protein expression levels of GRP78， CHOP， NF-κB p65， and COX-2 were significantly higher in OGD/R model group than those in control group （P<0.05）. When compared with OGD/R model group， the expression levels of GRP78， CHOP， NF-κBp65 and COX-2 were decreased by NaHS at concentrations of 0.2， 0.4 and 4 mmol/L （P<0.05）， but there was no statistically significant difference among the treatment groups （P>0.05）. 【Conclusion】 NaHS can rescue cell viability of OGD/R-induced cell injury. It may be achieved by inhibiting the activation of NF-κBp65 and COX-2 proteins induced by endoplasmic reticulum stress to reduce the level of cell apoptosis.

Fritillaria thunbergii Miq. has been widely used in traditional Chinese medicine for its expectorant, antitussive, antiinflammatory and analgesic properties. Moreover, modern pharmacological studies have demonstrated that F. thunbergii Miq. has efficacy in the treatment of leukemia and cancers of the liver and cervix. Although the alkaloid, peimine, is largely responsible for these pharmacological effects, it has very low oral bioavailability. The aim of this study was to investigate the intestinal absorption of peimine in Caco-2 cell monolayers. Having demonstrated that peimine is non-toxic to Caco-2 cells at concentrations <200 μmol/L, the effect of peimine concentration, pH, temperature, efflux transport protein inhibitors and EDTA-Na2 on peimine transport were studied. The results show that peimine transport is concentration-dependent; that at pH 6.0 and 7.4, the P app(AP-BL) of peimine is not significantly different but the P app(BL-AP)) is; that both P app(AP-BL) and P app(BL-AP) at 4 °C are significantly higher than their corresponding values at 37 °C; that the P-glycoprotein (P-gp) inhibitors, verapamil and cyclosporin A, increase absorption of peimine; and that EDTA-Na2 has no discernible effect. In summary, the results demonstrate that the intestinal absorption of peimine across Caco-2 cell monolayers involves active transport and that peimine is a substrate of P-gp.

Objective To investigate the effect of insulin-like growth factor 1 antibody (IGF-1Ab) on motor endplate function after injection ofbotulinum toxin A (Btx-A).Methods The total 90 male SD rats were randomly divided into 4 groups:control group,Btx-A group and 2 ug and 20 ug IGF-1Ab groups.In Btx-A and IGF-1Ab groups,a volume of 0.5 U (0.1 mL) Btx-A was intramuscularly injected into a site in the fight gastrocnemius muscle;on day 3,equal volumes (0.1 mL) of IGF-1Ab with a dosage of 2 ug and 20 ug were injected to the 2 ug and 20 ug IGF-1Ab groups respectively at the same site.The gastrocnemius muscle strength and the mean optical density (MOI) value of the positive reaction zone of acetylcholine esterase staining were evaluated at different time points.Results The gastrocnemius muscle strength increased from 12.34±0.16 g before injection to 7.70±0.90 g after injection in the Btx-A group;the gastrocnemius muscle strength decreased in other groups after injection of Btx-A;on day 14,28,42,56 and 70,the muscle strength oflGF-1Ab groups was significantly lower than that of Btx-A group (P＜0.05),and on day 42-70,the value of muscle strength of 20 ug IGF-1Ab group was signficantly lower than that of 2 μg IGF-1Ab group (P＜0.05).The MOI values of the positive reaction zone changed with the same trend.Conclusion IGF-1Ab can suppress the restore of motor endplate function after injection of Btx-A.

ABSTRACT:The aim of the present study was to investigate the inhibitory effects of TLR7 on Mycobacterium tuberculosis . TLR7 on infected RAW264 .7 cells was activated by chemical synthesis of TLR7 activation motif ssRNA .Activated RAW264 .7 cells were inoculated with Mycobacterium tuberculosis ,quantitative PCR method was applied to detect the phagocytosis rate of cell to bacteria at different time after infection .Cytokine production was measured by ELISA from cell supernatant .Cells were cultured on Roche medium and counted after sterile cracked with TritonX‐100 and diluted with PBS .Scanning electronic micro‐scope ( SEM ) was applied to detect the morphological changes of cells treated with TLR7 activation motif ssRNA .The highest phagocytosis rate of bacteria of RAW264 .7 cells was at 3 hours post infection (P>0 .05) .Compared with that of the control group ,treatment after 36 hours intracellular bacterial quantity in ssRNA treated group was lower (P<0 .05) ,levels of IL‐12 (P<0 .05) and IL‐4 (P<0 .05) were increased .For treatment after 48 hours ,level of IL‐4 (P<0 .05) was decreased ,and TNF‐α (P<0 .05) was increased .For treatment after 3 hours ,cell morphology of the ssRNA group was obviously better than the control group and appeared lots of phagosomes .Results suggested that TLR7 could enhance macrophages in killing Myco‐bacterium tuberculosis by forming phagosomes and regulating cytokines production ,and TLR7 activation motif ssRNA could be used in the treatment of tuberculosis .

Objective To dynamically monitor the effect of Cinepazide maleate (CM) on ischemic region of the brain and to elucidate the neuroprotection of CM on acute cerebral ischemic rat model and related mechanisms.Methods The rat model of acute brain ischemia-reperfusion was established with tighting threads.They were randomly divided into ischemia-reperfusion group and CM treatment group(n=8 in each group).Rats in CM treatment group were given 3 mg / kg CM by caudal intravenous injection immediately after brain ischemia-reperfusion.T2 weighted imaging(T2WI) and proton magnetic resonance spectroscopy (1HMRS) were performed before operation and at 6 hours,24 hours and 3 days after reperfusion.Results At 6 h,24 h and 3 d after operation there was no difference for areas of cerebral ischemia on T2WI between 2 groups.The values of N-acetylaspartate(NAA) (Phosphocreatine(PCr) +Creartine(Cr)) in ischemia-reperfusion group rats were (1.53±0.20),(0.50 ±0.17),(0.44±0.13),(0.40±0.10) before operation and at 6 h,24 h,3 d after operation,respectively.And in CM treatment group the values of NAA/(PCr+Cr) were (1.44±0.22),(0.68±0.13),(0.61±0.10),(0.53±0.09) at the same time points.The values of lactate(Lac)/(PCr+Cr) in ischemia-reperfusion group were (0.03±0.01),(1.10 ±0.28),(1.30± 0.23),(1.23± 0.19) before operation and at 6 h,24 h,3 d after operation,respectively.And in CM treatment group the values of Lac/(PCr+Cr) were (0.02±0.01),(0.85±0.25),(0.99±0.20),(0.90±0.15) at the same time points.The rats in ischemia-reperfusion group had lower value of NAA/(PCr+Cr) (P＜0.01) and higher value of Lac/(PCr+Cr) at all time points after operation than those before operation.Compared with rats in ischemia-reperfusion group,the value of NAA/(PCr+Cr) increased(P＜0.05) and the value of Lac/(PCr+Cr) decreased(P＜0.05) decreased significantly for CM treatment group rats.Conclusion CM treatment can decrease the intracellular accumulation of lactic acid and reduce neuronal necrosis in acute brain ischemia-reperfusion rats.