Objective To investigate the effects and mechanism of Ferrostatin-1(Fer-1),a ferroptosis inhibitor,on myocardial ischemia-reperfusion injury(MIRI).Methods Rat H9c2 cardiomyocytes were ran-domly divided into five groups:Control group,H/R medium group,H/R medium+Fer-1 group,H/R medium+Nec-1 group,and H/R medium+emricasan group.Cell morphology was observed using electron mi-croscopy.Cell proliferation activity was assessed via CCK-8 assay and lactate dehydrogenase(LDH)release.I-ron ion levels were measured using an iron detection kit.Reactive oxygen species(ROS)and mitochondrial su-peroxide levels were detected by flow cytometry and MitoSOXTM fluorescence staining,respectively.Western blot was employed to analyze the expression of glutathione peroxidase 4(GPX4),acyl-CoA synthetase long-chain family member 4(ACSL4),nicotinamide adenine dinucleotide phosphate oxidase(NOX1),and cycloox-ygenase 2(COX2).Results Compared to the Control group,the H/R medium group exhibited significantly increased cytotoxicity(LDH levels)and reduced cell viability,with statistically significant differences(P<0.05).Treatment with Fer-1,Nec-1,or emricasan in the H/R medium group increased cell adherence,reduced vacuolization,enhanced cell viability,and decreased cytotoxicity(LDH relative releasing rate)compared to the H/R medium group.Intracellular ferrous iron and total iron levels were elevated in the H/R medium group compared to the Control group,with statistically significant differences(P<0.05),while Fer-1 treatment sig-nificantly reduced these levels(P<0.05).ROS levels were higher in the H/R medium group than in the Con-trol group,and Fer-1 treatment attenuated this increase(P<0.05).Western blot analysis revealed elevated ACSL4,NOX1,and COX2 levels,alongside reduced GPX4 levels,in the H/R medium group compared to the Control group,with statistically significant differences(P<0.05).Fer-1 treatment reversed these trends,de-creasing ACSL4,NOX1,and COX2 levels while increasing GPX4 expression,with statistically significant differences(P<0.05).Conclusion Ferroptosis plays a critical role in MIRI.Fer-1 mitigates oxidative stress injury and alleviates MIRI by inhibiting ferroptosis.

[Objective]To investigate the mechanism action of Yiqi Bushen Formula on the proliferation and migration of gastric cancer stem cells(GCSCs).[Methods]SGC-7901 GCSCs with double positive markers of CD44 and epithelial cell adhesion molecule(EpCAM)were sorted by immunomagnetic beads.The selected CD44+EpCAM+SGC-7901 GCSCs were subcultured and stably transfected with circ_0051246 control,pcDNA circ_0051246 and si-circ_0051246 respectively.The above four groups of cells(including untransfected)were divided into three subgroups:blank control group,negative control group and Yiqi Bushen Formula group.The blank control group was normally subcultured,the negative control group was intervened with blank serum,and the Yiqi Bushen Formula group was intervened with Yiqi Bushen Formula-containing serum.The microstructure of SGC-7901 GCSCs was observed by laser confocal and transmission electron microscopy.Cell counting kit-8(CCK-8)and cell cloning experiments were used to detect the proliferation ability of SGC-7901 GCSCs.Flow cytometry was used to detect the positive rate of CD44 and EpCAM and the apoptosis rate.Cell scratch test and transwell were used to detect the migration and invasion ability of SGC-7901 GCSCs.The expression of circ_0051246 in each experimental group was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).[Results]Compared with respective negative control group,the number of mitochondrial cristae in each group of Yiqi Bushen Formula was significantly reduced,the endoplasmic reticulum was significantly swollen.Compared with respective blank control group,the expression of actin in the cytoskeleton structure of each group of Yiqi Bushen Formula was significantly decreased.Compared with respective negative control group,the cell proliferation ability of Yiqi Bushen Formula groups was weakened(P＜0.05,P＜0.01),the rate of double positive cells of CD44 and EpCAM antibodies was significantly decreased(P＜0.01),the cloning ability was significantly decreased(P＜0.01),the apoptosis rate was significantly increased(P＜0.01),the migration rate was significantly decreased at 24 and 48 h(P＜0.05,P＜0.01),the number of cell migration and invasion was significantly reduced(P＜0.01).The expression level of circ_0051246 in Yiqi Bushen Formula group was significantly decreased(P＜0.01).[Conclusion]Yiqi Bushen Formula may play a role in anti-proliferation and migration of GCSCs by inhibiting the expression of circ_0051246 and reducing the expression of SGC-7901 CSCs surface markers CD44 and EpCAM,which provides a new idea for the treatment of gastric cancer.

[Objective]To investigate the mechanism action of Yiqi Bushen Formula on the proliferation and migration of gastric cancer stem cells(GCSCs).[Methods]SGC-7901 GCSCs with double positive markers of CD44 and epithelial cell adhesion molecule(EpCAM)were sorted by immunomagnetic beads.The selected CD44+EpCAM+SGC-7901 GCSCs were subcultured and stably transfected with circ_0051246 control,pcDNA circ_0051246 and si-circ_0051246 respectively.The above four groups of cells(including untransfected)were divided into three subgroups:blank control group,negative control group and Yiqi Bushen Formula group.The blank control group was normally subcultured,the negative control group was intervened with blank serum,and the Yiqi Bushen Formula group was intervened with Yiqi Bushen Formula-containing serum.The microstructure of SGC-7901 GCSCs was observed by laser confocal and transmission electron microscopy.Cell counting kit-8(CCK-8)and cell cloning experiments were used to detect the proliferation ability of SGC-7901 GCSCs.Flow cytometry was used to detect the positive rate of CD44 and EpCAM and the apoptosis rate.Cell scratch test and transwell were used to detect the migration and invasion ability of SGC-7901 GCSCs.The expression of circ_0051246 in each experimental group was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).[Results]Compared with respective negative control group,the number of mitochondrial cristae in each group of Yiqi Bushen Formula was significantly reduced,the endoplasmic reticulum was significantly swollen.Compared with respective blank control group,the expression of actin in the cytoskeleton structure of each group of Yiqi Bushen Formula was significantly decreased.Compared with respective negative control group,the cell proliferation ability of Yiqi Bushen Formula groups was weakened(P＜0.05,P＜0.01),the rate of double positive cells of CD44 and EpCAM antibodies was significantly decreased(P＜0.01),the cloning ability was significantly decreased(P＜0.01),the apoptosis rate was significantly increased(P＜0.01),the migration rate was significantly decreased at 24 and 48 h(P＜0.05,P＜0.01),the number of cell migration and invasion was significantly reduced(P＜0.01).The expression level of circ_0051246 in Yiqi Bushen Formula group was significantly decreased(P＜0.01).[Conclusion]Yiqi Bushen Formula may play a role in anti-proliferation and migration of GCSCs by inhibiting the expression of circ_0051246 and reducing the expression of SGC-7901 CSCs surface markers CD44 and EpCAM,which provides a new idea for the treatment of gastric cancer.

Objective To investigate the effect of perineal nerve electroacupuncture combined with tolterodine in the treatment of female overactive bladder(OAB).Methods A total of 150 female OAB patients treated in Huzhou Hospital of Traditional Chinese Medicine,Zhejiang Chinese Medical University from April 2020 to May 2021 were selected and divided into control group and study group according to random number table method,with 75 cases in each group.The control group was treated with tolterodine,and the study group was treated with pudendal nerve electroacupuncture combined with tolterodine.Clinical symptoms,urinary symptoms,and urodynamic indicators[maximum cystometric capacity(MCC),maximum urethral closure pressure(Pura clos max),maximum detrusor pressure(Pdet max),and maximum urine flow rate(Qmax)],bladder function indicators(initial bladder sensation volume,bladder volume during strong micturition desire),nerve growth factor(NGF)mediated transient receptor potential(TRP)pathway indicators[NGF,NGF/urine creatinine(UCr),TRPV1,TRPV4]and clinical efficacy of two groups were compared.Results After treatment,the number of night urination and urgent urination in study group were significantly less than those in control group,and the average volume of urination was significantly more than that in control group(P＜0.05).The overactive bladder symptom scores and visual analogue scale scores of study group were significantly lower than those of control group(P＜0.05).The MCC and Pura·clos·max in study group were significantly higher than those in control group,while Pdet max and Qmax were significantly lower than those in control group(P＜0.05).The initial bladder sensation volume and bladder volume during strong micturition desire in study group were higher than those in control group(P＜0.05).NGF,NGF/UCr,positive expression rates of TRPV1 and TRPV4 in study group were lower than those in control group(P＜0.05).The total effective rate was significantly higher in study group than in control group,(x2=5.374,P=0.020).There was no significant difference in the incidence of total adverse reactions between two groups(x2=0.362,P=0.547).Conclusion Perineal nerve electroacupuncture combined with tolterodine can significantly improve bladder function and inhibit clinical symptoms in female OAB patients,possibly by inhibiting bladder sensitivity caused by NGF-mediated increase of TRP channel proteins TRPV1 and TRPV4.

Objective:To analyze the clinical data and genetic characteristics of a child with CLN1 neuronal ceroid lipofuscinosis in conjunct with hereditary hyperferritinemia cataract syndrome (HHCS).Methods:A child who was admitted to the PICU of the First Affiliated Hospital of Zhengzhou University in November 2020 was selected as the study subject. Clinical data of the child was collected. Genetic testing was carried out for the child, and the result was analyzed in the light of literature review to explore the clinical and genetic characteristics to facilitate early identification.Results:The patient, a 3-year-old male, had mainly presented with visual impairment, progressive cognitive and motor regression, and epilepsy. Cranial magnetic resonance imaging revealed deepened sulci in bilateral cerebral hemispheres, and delayed myelination. The activity of palmitoyl protein thioesterase was low (8.4 nmol/g/min, reference range: 132.2 ~ 301.4 nmol/g/min), whilst serum ferritin was increased (2 417.70 ng/mL, reference range: 30 ~ 400 ng/mL). Fundoscopy has revealed retinal pigment degeneration. Whole exome sequencing revealed that he has harbored c. 280A>C and c. 124-124+ 3delG compound heterozygous variants of the PPT1 gene, which were respectively inherited from his father and mother. Neither variant has been reported previously. The child has also harbored a heterozygous c. -160A>G variant of the FTL gene, which was inherited from his father. Based on the clinical phenotype and results of genetic testing, the child was diagnosed as CLN1 and HHCS. Conclusion:The compound heterozygous variants of the PPT1 gene probably underlay the disorders in this child. For children with CLN1 and rapidly progressing visual impairment, ophthalmological examination should be recommended, and detailed family history should be taken For those suspected for HHCS, genetic testing should be performed to confirm the diagnosis.

Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective To investigate the predictive value of white blood cell-to-hematocrit ratio (WBCHR) for in-hospital major adverse cardiovascular events (MACE) after reperfusion therapy in patients with acute ST-segment elevation myocardial infarction (STEMI). Methods A case-control study was conducted to retrospectively select 319 patients with first-time diagnosis of STEMI who underwent percutaneous coronary intervention (PCI). Patients were divided into MACE group (69 cases) and non-MACE group (250 cases) based on the occurrence of MACE during hospitalization. Clinical data, including general information, laboratory test indicators, echocardiography, and coronary angiography results, were compared between the two groups. Univariate and multivariate Logistic regression analyses were performed to explore the risk factors for in-hospital MACE after reperfusion therapy in STEMI patients. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the predictive value of WBCHR for in-hospital MACE after reperfusion therapy in STEMI patients. Results The levels of fasting blood glucose, uric acid, creatinine, white blood cell count, neutrophil count, high-sensitivity C-reactive protein (hs-CRP), D-dimer, and WBCHR were significantly higher in the MACE group than in the non-MACE group, while red blood cell count, hemoglobin, hematocrit, and left ventricular ejection fraction were lower (P < 0.05). No significant differences were observed in age, gender, smoking history, hypertension history, diabetes history, door-to-wire (D2W) time, total cholesterol, triglycerides, and the number of diseased vessels between the two groups (P>0.05). Multivariate Logistic regression analysis revealed that elevated uric acid levels (OR=1.005; 95%CI, 1.002 to 1.009; P=0.004), decreased hemoglobin concentration (OR=0.964; 95%CI, 0.941 to 0.988; P=0.003), increased hs-CRP levels (OR=1.032; 95%CI, 1.009 to 1.056; P=0.007), and increased WBCHR (OR=1.455; 95%CI, 1.295 to 1.635; P < 0.001) were independent risk factors for in-hospital MACE after reperfusion therapy in STEMI patients. ROC curve analysis showed that the area under the curve of WBCHR for predicting in-hospital MACE after reperfusion therapy in STEMI patients was 0.855 (95%CI, 0.796 to 0.914, P < 0.001), with a sensitivity of 63.8% and a specificity of 99.6%. Conclusion WBCHR is an independent influencing factors of in-hospital MACE after reperfusion therapy in STEMI patients and has a high predictive value for in-hospital MACE in these patients.

Objective:To analyze the effect of perineural invasion(PNI)on immune cell infiltration in pancreatic ductal adenocarcinoma(PDAC)by the CIBERSORT deconvolution algorithm.Methods:The pancreatic cancer patients from the dataset GSE102238 were re-evaluated for the severity of PNI.And then the high and low PNI subgroups were subjected to immune scoring and immune cell infiltration analysis using the ESTIMATE and CIBERSORT algorithms to find immune cell subgroups associated with PNI.Finally,the results were validated by tissue microarrays.Results:Twenty-five cases were selected from 50 pancreatic cancer specimens for PNI reassessment and then divided into two high and low groups.Compared to the low PNI group,specimens from patients in the high group showed significantly less CD8+T-cell infiltration(P<0.05)and significantly more resting memory CD4+T-cells and M0 macrophages(P<0.05).Significantly reduced CD8+T cells(P<0.01)and slightly increased CD4+T cells(P<0.05)were confirmed in the patients with the high PNI using tissue microarrays.Meanwhile,macrophages significantly increased in the high PNI group(P<0.001).Conclusion:High PNI in PDAC inhibits infiltration of CD8+T cells which promote the infiltration of macrophages.

Objective:To analyze the effect of perineural invasion(PNI)on immune cell infiltration in pancreatic ductal adenocarcinoma(PDAC)by the CIBERSORT deconvolution algorithm.Methods:The pancreatic cancer patients from the dataset GSE102238 were re-evaluated for the severity of PNI.And then the high and low PNI subgroups were subjected to immune scoring and immune cell infiltration analysis using the ESTIMATE and CIBERSORT algorithms to find immune cell subgroups associated with PNI.Finally,the results were validated by tissue microarrays.Results:Twenty-five cases were selected from 50 pancreatic cancer specimens for PNI reassessment and then divided into two high and low groups.Compared to the low PNI group,specimens from patients in the high group showed significantly less CD8+T-cell infiltration(P<0.05)and significantly more resting memory CD4+T-cells and M0 macrophages(P<0.05).Significantly reduced CD8+T cells(P<0.01)and slightly increased CD4+T cells(P<0.05)were confirmed in the patients with the high PNI using tissue microarrays.Meanwhile,macrophages significantly increased in the high PNI group(P<0.001).Conclusion:High PNI in PDAC inhibits infiltration of CD8+T cells which promote the infiltration of macrophages.

Objective:To investigate the relationship between the E2 and E4 alleles of apolipoprotein E (apoE) gene and myocardial infarction (MI) in type 2 diabetes Mellitus (T2DM) patients, and to explore the relationship between apoE polymorphism and blood lipid metabolism.Methods:This case control study was conducted from August 2016 to March 2020 in China-Japan Friendship Hospital, 3 459 inpatients with T2DM were included including 3 044 patients without MI (T2DM group) and 415 patients with MI (T2DM+MI group). Real time fluorescent quantitative PCR was used to detect apoE polymorphism. Automatic biochemical analyzer was used to detect lipid levels. Logistic regression analyses were performed to determine the association of apoE with risk of MI in patients with T2DM.Results:(1) The frequency of E4 allele in T2DM+MI group (12.29%, 102/830) was significantly higher than in T2DM group (9.13%,556/6 088), while the frequency of E2 allele in T2DM+MI group (7.35%,61/830) was significantly lower than that in T2DM group (8.21%,500/6 088), P=0.012. Logistic regression analyses showed that E4 allele carrier (E3/E4+E4/E4) faced a higher risk for MI in T2DM patients ( OR=1.48, 95% CI 1.14-1.92, P=0.003), while E2 allele carrier(E2/E3+E2/E2)did not face a higher risk of MI in T2DM patients ( OR=0.88, P=0.642). (2) The levels of apoE polymorphism and blood lipid: The levels of TC, LDL-C and apoB increased in the order of E4 allele, wild type and E2 allele ( P<0.05). The levels of HDL-C, apoA1 and apoE decreased in the order of E4 allele, Wild type and E2 allele ( P<0.05). Conclusion:The E4 allele is a risk factor for MI in T2DM patients, and apoE polymorphism can affect blood lipid level in this patent cohort.