BACKGROUND: Concurrent chemo-radiotherapy (CCRT) is the standard treatment for locally advanced cervical cancer (LACC), but there are still many patients who suffer tumor recurrence. However, valuable predictors of treatment outcomes remain limited. This study aimed to assess the value of the serum immune biomarkers to predict the prognosis. METHODS: We reviewed cervical cancer patients treated with CCRT between January 2014 and May 2018 at Peking Union Medical College Hospital. The systemic immune inflammation index (SII), systemic inflammation response index (SIRI), and lactate dehydrogenase (LDH) were calculated using blood samples. The relationship between immune markers and the treatment outcome was analyzed. The area under the receiver operating characteristic (ROC) curve was used to evaluate the predictive efficiency. The Cox proportional hazards model and log-rank were used to predict overall survival (OS) and disease-free survival (DFS). RESULTS: This study included 667 patients. Among them, 195 (29.2%) patients were defined as treatment failure, including 127 (19.0%) patients with pelvic failure, 94 (14.1%) distant failure, and 25 (3.7%) concurrent pelvic and distant failure. It revealed that the tumor stage, size, metastatic lymph nodes (MLNs), and serum immune biomarkers, such as SII, SIRI, and LDH, were significantly related to treatment outcomes. We demonstrated that the optimal cut-off of the SII, SIRI, and LDH were 970.4 × 10 9 /L, 1.3 × 10 9 /L, and 207.52 U/L, respectively. Importantly, this study presented that LDH level had the highest OR (OR = 4.2; 95% CI [2.3-10.8]). Furthermore, the OS and DFS for patients with pre-SII ≥970.5 × 10 9 /L were significantly worse than those with pre-SII <970.5 × 10 9 /L. Similarly, pre-SIRI ≥1.25 × 10 9 /L and pre-LDH ≥207.5 U/L were related to poor survival outcomes. CONCLUSIONS This study demonstrated that the baseline SII, SIRI, and LDH levels can be used to accurately and effectively predict the treatment outcomes after CCRT and long-term prognosis. Our results may offer additional prognostic information in clinical, which helps to detect the potential recurrent metastasis in time.
Humans ; Female ; Uterine Cervical Neoplasms/drug therapy* ; Middle Aged ; Adult ; Aged ; Chemoradiotherapy/methods* ; L-Lactate Dehydrogenase/blood* ; Treatment Outcome ; Disease-Free Survival ; Prognosis ; ROC Curve ; Biomarkers, Tumor/blood* ; Proportional Hazards Models

OBJECTIVES: To investigate the risk factors for plastic bronchitis (PB) in children with macrolide-unresponsive Mycoplasma pneumoniae pneumonia (MUMPP) and to establish a nomogram prediction model. METHODS: A retrospective analysis was conducted on 178 children with MUMPP who underwent bronchoscopy from January to December 2023. According to the presence or absence of PB, the children were divided into a PB group (49 children) and a non-PB group (129 children). The predictive factors for the development of PB in children with MUMPP were analyzed, and a nomogram prediction model was established. The model was assessed in terms of discriminatory ability, accuracy, and clinical effectiveness. RESULTS: The multivariate logistic regression analysis showed that older age and higher levels of lactate dehydrogenase and fibrinogen were closely associated with the development of PB in children with MUMPP (P<0.05). A nomogram model established based on these factors had an area under the receiver operating characteristic curve of 0.733 (95%CI: 0.651-0.816, P<0.001) and showed a good discriminatory ability. The Hosmer-Lemeshow goodness-of-fit test indicated that the predictive model had a good degree of fit (P>0.05), and the decision curve analysis showed that the model had a good clinical application value. CONCLUSIONS The risk nomogram model established based on age and lactate dehydrogenase and fibrinogen levels has good discriminatory ability, accuracy, and predictive efficacy for predicting the development of PB in children with MUMPP.
Retrospective Studies ; Risk Factors ; Nomograms ; Mycoplasma pneumoniae/isolation & purification* ; Pneumonia, Mycoplasma/microbiology* ; Bronchitis/microbiology* ; Macrolides/therapeutic use* ; Drug Resistance, Bacterial ; Bronchoscopy ; Area Under Curve ; ROC Curve ; Fibrinogen/analysis* ; Age Factors ; Humans ; Male ; Female ; Infant ; Child, Preschool ; Child ; Adolescent ; L-Lactate Dehydrogenase/blood*

OBJECTIVE: To explore the role of reactive oxygen species (ROS) and lactate dehydrogenase isoenzyme X (LDH-X) in the changes of sperm mitochondrial membrane potential (MMP) in infertility patients with varicocele (VC). METHODS: This study included 38 infertility patients with VC (VCinf), 35 non-VC infertile males (NVCinf), and 30 normal fertile men as controls. We obtained the routine semen parameters using the sperm quality analysis system, examined the contents of LDH-X in the seminal plasma and sperm with the automatic biochemical analyzer, measured the level of malondialdehyde (MDA) in seminal plasma by thiobarbituric acid (TBA) colorimetry, and determined the expressions of mitochondrial membrane potential (MMP) and LDH-X mRNA in the sperm using JC-1 fluorescence probe and RT-PCR. RESULTS: No statistically significant differences were observed among the three groups of subjects in age, semen pH value, semen volume and sperm concentration (P > 0.05). Compared with the normal fertile controls, the patients in the VCinf and NVCinf groups showed significantly decreased sperm motility (［52.36 ± 12.48］% vs ［34.74 ± 15.23］% vs ［25.76 ± 13.73］%, P< 0.05), percentage of progressively motile sperm (PMS) (［42.54 ± 13.58］% vs ［29.10 ± 14.17］% vs ［20.95 ± 12.33］%, P< 0.05), sperm LDH-X (［16.46 ± 5.47］ vs ［13.63 ± 4.50］ vs ［10.18 ± 3.00］ mU/106, P< 0.05), sperm MMP (［48.04 ± 11.62］% vs ［40.86 ± 12.69］% vs ［34.41 ± 13.93］%, P< 0.05) and expression of sperm LDH-X mRNA (P< 0.05). but increased seminal plasma LDH-X (［935.36 ± 229.48］ vs ［1241.05 ± 337.07］ vs ［1425.08 ± 469.35］ U/L, P< 0.05), seminal plasma/whole sperm LDH-X (［1.06 ± 0.35］ vs ［1.40 ± 0.34］ vs ［1.63 ± 0.66］, P< 0.05), and content of seminal plasma MDA (［1.10 ± 0.19］ vs ［1.59 ± 0.27］ vs ［2.00 ± 0.22］ nmol/ml, P< 0.05). CONCLUSION Excessive ROS in the reproductive system of VCinf patients reduces the content of MMP and causes the overflow of LDH-X out of sperm cells. Therefore the decrease of sperm LDH-X may be accompanied by that of MMP.
Humans ; Male ; Infertility, Male/etiology* ; Varicocele/metabolism* ; Adult ; Reactive Oxygen Species/metabolism* ; Spermatozoa/metabolism* ; L-Lactate Dehydrogenase/metabolism* ; Membrane Potential, Mitochondrial ; Isoenzymes/metabolism* ; Case-Control Studies ; Young Adult ; Mitochondrial Membranes/metabolism*

The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Objective: To investigate the predictive factors for bronchitis obliterans in refractory Mycoplasma pneumoniae pneumonia (RMPP). Methods: A restrospective case summary was conducted 230 patients with RMPP admitted to the Department of No.2 Respiratory Medicine of Beijing Children's Hospital, Capital Medical University from January 2013 to June 2017 were recruited. Clinical data, laboratory results, imaging results and follow-up data were collected. Based on bronchoscopy and imaging findings 1 year after discharge, all patients were divided into two groups: one group had sequelae of bronchitis obliterans (sequelae group) and the other group had not bronchitis obliterans (control group), independent sample t-test and nonparametric test were used to compare the differences in clinical features between the two groups. Receiver operating characteristic (ROC) curve to explore the predictive value of Bronchitis Obliterans in RMPP. Results: Among 230 RMPP children, there were 115 males and 115 females, 95 cases had sequelae group, the age of disease onset was (7.1±2.8) years;135 cases had control group, the age of disease onset was (6.8±2.7) years. The duration of fever, C-reative protein (CRP) and lactate dehydrogenase (LDH) levels, the proportion of ≥2/3 lobe consolidation, pleural effusion and the proportion of airway mucus plug and mucosal necrosis were longer or higher in the sequelae group than those in the control group ((17±9) vs. (12±3) d, (193±59) vs. (98±42) mg/L,730 (660, 814) vs. 486 (452, 522) U/L, 89 cases (93.7%) vs. 73 cases (54.1%), 73 cases (76.8%) vs.59 cases (43.7%), 81 cases (85.3%) vs. 20 cases (14.8%), 67 cases (70.5%) vs. 9 cases (6.7%), t=5.76, 13.35, Z=-6.41, χ²=14.64, 25.04, 22.85, 102.78, all P<0.001). Multivariate Logistic regression analysis showed that the duration of fever ≥10 days (OR=1.200, 95%CI 1.014-1.419), CRP levels increased (OR=1.033, 95%CI 1.022-1.044) and LDH levels increased (OR=1.001, 95%CI 1.000-1.003) were the risk factors for sequelae of bronchitis obliterans in RMPP. ROC curve analysis showed that CRP 137 mg/L had a sensitivity of 82.1% and a specificity of 80.1%; LDH 471 U/L had a sensitivity of 62.7% and a specificity of 60.3% for predicting the development of bronchitis obliterans. Conclusions: The long duration of fever (≥10 d), CRP increase (≥137 mg/L) may be used to predict the occurrence of sequelae of bronchitis obliterans in RMPP. It is helpful for early recognition of risk children.
Child ; Male ; Female ; Humans ; Child, Preschool ; Mycoplasma pneumoniae ; Retrospective Studies ; Pneumonia, Mycoplasma/complications* ; Disease Progression ; L-Lactate Dehydrogenase ; Fever

Objective: To explore the value of paraquat (PQ) intake, urine protein and myocardial enzyme indexes in judging the prognosis of patients with acute PQ poisoning. Methods: From September to December 2021, all 201 patients with acute PQ poisoning admitted to Guangzhou Twelfth People's Hospital from January 2010 to December 2019 were selected as the research objects. Based on follow-up results 60 days after poisoning, the research objects were divided into survival group (n=78) and death group (n=123) . The differences in information about poisoning, treatment plan, PQ intake, urine protein, creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase between the two groups of patients were compared and analyzed. Logistic regression and Cox regression were used to analyze the correlation between poisoning outcome and PQ intake, urine protein and myocardial enzymes. ROC curve and principal component analysis were used to explore high-efficiency indicators for predicting the outcome of acute PQ poisoning. Results: The PQ intake[50 (20, 100) ml], urine protein (total rank 15570.50) , creatine kinase[ (336.36±261.96) U/L], creatine kinase isoenzyme[ (43.91±43.74) U/L], lactate dehydrogenase [ (346.01±196.50) U/L], α-hydroxybutyrate dehydrogenase content[ (271.23±11.92) U/L] of patients in the death group were all higher than the survival group[15 (10, 20) ml, 4730.50, (187.78±178.06) U/L, (18.88±15.50) U/L, (190.92±60.50) U/L, (152.60±48.34) U/L, respectively] (P<0.05) . The outcome of acute PQ poisoning was positively correlated with PQ intake, urine protein, creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase (P<0.05) . Multivariate logistic regression and multivariate Cox regression analysis showed that creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase and α-hydroxybutyrate dehydrogenase was positively correlated with the prognosis of patients with acute PQ poisoning (P<0.05) . ROC curve analysis and principal component analysis showed that the combined indexes of PQ intake, urine protein and myocardial enzymes had the highest efficacy and weight in judging the prognosis of patients (AUC=0.91, weight coefficient=0.19, sensitivity=0.76, specificity=0.89) . When the combined score was ≥4, the probability of accurately predicting the death of patients was as high as 91% (positive predictive value=0.91) . Conclusion: PQ intake, urine protein combined with creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase has high value in predicting the prognosis of patients with acute PQ poisoning.
Humans ; Creatine ; Creatine Kinase ; Isoenzymes ; Lactate Dehydrogenases ; Paraquat/poisoning* ; Prognosis ; Retrospective Studies ; Myocardium/enzymology* ; Urine/chemistry*

OBJECTIVE: To analyze the expression and correlation of microRNA-195 (miR-195), miR-125 and calreticulin in diffuse large B-cell lymphoma (DLBCL). METHODS: From April 2020 to April 2021, 80 DLBCL patients with complete data archived by the Pathology Department of Handan First Hospital and The Second Hospital of Hebei Medical University were selected as the study group, and 70 patients with reactive lymph node hyperplasia were selected as the control group. The expressions of miR-195 and miR-125 were detected by real-time fluorescence quantitative PCR, and the expression of calreticulin was detected by Western blot. Pearson correlation was used to analyze the correlation between miR-195, miR-125, calreticulin and DLBCL, and ROC curve was used to analyze the predictive value of miR-195, miR-125 and calreticulin for DLBCL. RESULTS: Compared with the control group, the expression of miR-195 decreased but miR-125 and calreticulin increased in the study group (P<0.001). The expression levels of miR-195, miR-125 and calreticulin were not related to sex, age, primary site and B symptoms of patients with DLBCL, but related to immunophenotype, Ann Arbor stage, lactate dehydrogenase, IPI score, nodule involvement and Ki-67 index. The expression of miR-195 decreased and the expression of miR-125 and calreticulin increased in DLBCL paitents with non-germinal center source, Ann Arbor stage III-IV, lactate dehydrogenase > 245 U/L, IPI score 3-5, nodule involvement≥2 and Ki-67 index≥75% (P<0.05). Pearson correlation analysis showed that miR-195 and miR-125 were negatively correlated (r=-0.536, P=0.001), miR-195 and calreticulin were negatively correlated (r=-0.545, P=0.001), while miR-125 and calreticulin were positively correlated (r=0.523, P=0.001). ROC curve showed that compared with the single diagnosis of miR-195, miR-125 and calreticulin, the combination of the three items had higher predictive value for DLBCL (P<0.001). CONCLUSION The expression of miR-195 decreases and the expression of miR-125 and calreticulin increase in patients with DLBCL. Along with the increase of disease stage and IPI score, the decrease of miR-195 and the increase of miR-125 and calreticulin aggravate gradually. The three items may participate in the occurrence and progress of DLBCL.
Humans ; MicroRNAs/genetics* ; Ki-67 Antigen/metabolism* ; Calreticulin/metabolism* ; Prognosis ; Lymphoma, Large B-Cell, Diffuse/genetics* ; Lactate Dehydrogenases/metabolism*

OBJECTIVE: To analyze the clinical characteristics of the patients with B-cell chronic lymphoproliferative disease（B-CLPD） in the new drug era and the effect of new drug treatment on efficacy and survival. METHODS: The clinical and laboratory data of 200 cases B-CLPD patients diagnosed between April 2015 and August 2021 were analyzed retrospectively. The clinical efficacy and survival of the patients under different treatments including Bruton tyrosine kinase（BTK） inhibitors， rituximab， and chemotherapy alone were analyzed. The prognostic factors affecting the survival of patients were analyzed by univarite analysis and multivariate analysis. RESULTS: There were 119 male（59.5％） and 81 female（40.5%） in 200 cases B-CLPD patients， the sex ratio(male/female) was 1.5∶1 with median age of 61（30- 91） years old. The distribution of subtypes were as fallows: 51 cases (25.5%) of chronic lymphocytic leukemia/small lymphocytic lymphoma（CLL/SLL）， 64（32.0%） cases of follicular lymphoma（FL）， 40（20.0%） cases mantle cell lymphoma（MCL）， 30（15.0%） cases of marginal zone lymphoma（MZL）， 10（5%） cases of lymphoplasmacytic lymphoma/waldenstrom macroglobulinemia（LPL/WM）， 5（2.5%） cases of B cell chronic lymphoproliferative disorders unclassified（B-CLPD-U） . The main clinical manifestation of 102 patients was lymph node enlargement, 32 cases were complicated with B symptoms. Among CLL/SLL patients， there were 12（23.5%） cases in Binet A and 39（76.5%） cases in Binet B/C. There were 29 patients（20.9%） in Ann Arbor or Lugano stage I-II and 110 cases（79.1%） in stage III-IV of other subtypes. The complete remission（CR） rate was 43.1%（25/58）， 40.2%（39/97）， 7.1%（1/14）， and overaIl response rate（ORR） was 87.9%（51/58）， 62.9%（61/97）， 28.6%（4/14） in the groups of BTK inhibitors， rituximab-based therapy， and chemotherapy alone. The 3-year OS rate and PFS rate in all patients was 79.2% and 72.4% respectively. The 3-year OS rate of patient with MZL, CLL/SLL, FL,WM was 94.7%, 87.7%, 86.8% and 83.3% respectively, while the 3-year OS rate of MCL was only 40.6%, which was significantly lower than other subtypes. The median OS of patients treated with BTK inhibitors and rituximab-based therapy was 20.5 and 18.5 months respectively， and the 3-year OS rate was 97.4% and 90.7%. However， the median PFS of patients receiving chemotherapy alone was 4 months， and the 1-year OS rate was 52.7%， which was statistically significant compared with the other two groups（P<0.05）. Univarite analysis showed that anemia， elevated lactate dehydrogenase， elevated β2-microglobulin， and splenomegaly were the poor prognostic factors for OS（P<0.05）， elevated lactate dehydrogenase was also poor prognostic factors for PFS（P<0.05）. Multifactor analysis showed that anemia and elevated lactate dehydrogenase were the independent poor prognostic factors for survival（P<0.05）. CONCLUSION The clinical features of B-CLPD was various， anemia and elevated lactate dehydrogenase are the prognostic factors for poor survival. BTK inhibitors and new immunotherapy can improve the survival and prognosis of patients in the new drug era.
Humans ; Adult ; Female ; Male ; Middle Aged ; Aged ; Aged, 80 and over ; Rituximab/therapeutic use* ; Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy* ; Retrospective Studies ; Lymphoma, Mantle-Cell ; Prognosis ; Lymphoma, B-Cell, Marginal Zone ; Lactate Dehydrogenases

OBJECTIVE: To analyze the effect of CD56 expression on the prognosis of newly diagnosed multiple myeloma (MM) patients and explore the relationship between CD56 with clinical characteristics. METHODS: In this retrospective study, the clinical data and laboratory parameters of 175 newly diagnosed MM patients from February 2015 to December 2020 in the Second Hospital of Anhui Medical University were collected. The patients were divided into CD56⁺ and CD56^- groups based on the expression of CD56, and the general data and laboratory parameters of the two groups were compared. The patients were followed up to June 30, 2021, and progression-free survival (PFS) and overall survival (OS) were recorded. PFS and OS curves of the two groups were plotted respectively, and the survival differences were compared. Univariate and multivariate Cox regression analyses were performed to analyze the effect of CD56 on the prognosis of newly diagnosed MM patients. RESULTS: In 175 newly diagnosed MM patients, 57(32.6%) cases were in the CD56^-group and 118 (67.4%) cases in the CD56⁺ group. There was significant correlation between CD56 expression and ISS stage, ECOG score, platelets, β₂-microglobulin, creatinine, and extramedullary disease (all P <0.05). The incidence of extramedullary disease in the CD56^- group was significantly higher than that in the CD56⁺ group (29.8% vs 12.7%, P =0.006). The median follow-up time of the whole cohort was 23.6 (1.0-78.6) months. The median PFS of patients in CD56⁺ group and CD56^- group were 18.6 (1.2-77.6) and 12.2 (1.0-49.0) months, respectively, and the median OS of the two groups were 27.6 (1.4-77.7) and 19.7 (1.0-78.6) months, respectively. The 2-year PFS rate in the CD56⁺ group was significantly higher than that in the CD56^- group (57.6% vs 36.8%, P =0.010), and the 2-year OS rate in the CD56⁺ group was higher than that in the CD56^- group, but it didn't reach statistical significance (74.6% vs 64.9%, P =0.158). The results of univariate Cox regression analysis showed that the PFS was significantly shorter in newly diagnosed MM patients with advanced age, type IgG, high ECOG score, decreased platelet count, increased lactate dehydrogenase level, extramedullary disease, and CD56^- (all P <0.05), the OS was significantly shorter in patients with high ECOG score, decreased platelet count, increased lactate dehydrogenase level, extramedullary disease, and CD56^- (all P <0.05). The results of multivariate Cox regression analysis showed that advanced age, type IgG, elevated lactate dehydrogenase level, extramedullary disease, and CD56^- were independent prognostic factors for poor PFS (all P <0.05); and decreased platelet count, elevated lactate dehydrogenase level, and extramedullary disease were independent adverse prognostic factors for OS (all P <0.05), while there was no significant independent correlation between CD56 and OS (P >0.05). CONCLUSION Most of the newly diagnosed MM patients have positive expression of CD56. Loss of CD56 expression was associated with unfavorable biological and clinical parameters and poor prognosis, suggesting that CD56 has important clinical value in the prognosis of newly diagnosed MM patients.
Humans ; Immunoglobulin G ; Lactate Dehydrogenases ; Multiple Myeloma/diagnosis* ; Prognosis ; Retrospective Studies

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases