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

This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

Objective To precisely identify the para-Bombay blood types in cancer patients at our hospital, establish a robust system for the identification of challenging blood types in our laboratory, and provide a foundation for precise transfusion practices. Methods We retrospectively analyzed the blood type results of 91 874 cancer patients from January 1, 2019, to December 31, 2023. Conventional serological methods were used to screen for blood types, and suspected para-Bombay blood types were identified. Further analysis was performed using Pacific Biosciences (PacBio) single-molecule real-time sequencing and Sanger sequencing was used to determine the genotypes of the ABO, FUT1, and FUT2 genes. Results Eight cases of para-Bombay blood type were confirmed through serological and molecular biological methods. The FUT1 genotypes identified were: 5 cases of h1h1 (homozygous mutation 551_552delAG) and 3 cases of h1h2 (compound heterozygous mutations of 551_552delAG and 880_882delTT). The FUT2 genotypes identified were: 2 cases of Se³⁵⁷/Se^{357, 716} and 4 cases of Se³⁵⁷/Se³⁵⁷. Additionally, one sample revealed a novel heterozygous mutation, 818C>T, in exon 7 of the ABO gene, which was confirmed by PacBio sequencing to be located on the O haplotype. Conclusion PacBio sequencing technology demonstrates significant advantages in analyzing the haplotypes of para-Bombay blood type genes. This approach supports the establishment of a robust system for the identification of challenging blood types and provides novel evidence for precise transfusion practices in cancer patients.
Humans ; Neoplasms/genetics* ; Fucosyltransferases/genetics* ; ABO Blood-Group System/genetics* ; Male ; High-Throughput Nucleotide Sequencing/methods* ; Galactoside 2-alpha-L-fucosyltransferase ; Female ; Retrospective Studies ; Genotype ; Middle Aged ; Blood Grouping and Crossmatching/methods* ; Adult ; Mutation ; Aged

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*

OBJECTIVE: To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca²⁺ channel (LTCC) and related signaling pathway. METHODS: Isometric tension recording was performed in HUV rings pre-contracted with K⁺. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current. RESULTS: After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104). CONCLUSIONS Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.
Hesperidin/pharmacology* ; Humans ; Calcium Channels, L-Type/metabolism* ; Umbilical Veins/physiology* ; Muscle Contraction/drug effects* ; Animals ; Rats ; Calcium Channel Blockers/pharmacology* ; Vasodilation/drug effects* ; Muscle Relaxation/drug effects*

OBJECTIVES: To investigate the effects of quercetin on cuproptosis and L-type calcium currents in the myocardium of diabetic rats. METHODS: Forty SD rats were randomized into control group and diabetic model groups. The rat models of diabetes mellitus (DM) induced by high-fat and high-sugar diet combined with streptozotocin (STZ) injection were further divided into DM model group, quercetin treatment group, and empagliflozin treatment group (n=10). Blood glucose and body weight were measured every other week, and cardiac function of the rats was evaluated using echocardiography. HE staining, Sirius red staining, and wheat germ agglutinin (WGA) analysis were used to observe the changes in myocardial histomorphology, and serum copper levels and myocardial FDX1 expression were detected. In cultured rat cardiomyocyte H9c2 cells with high-glucose exposure, the effects of quercetin and elesclomol, alone or in combination, on intracellular CK-MB and LDH levels and FDX1 expression were assessed, and the changes in L-type calcium currents were analyzed using patch-clamp technique. RESULTS: The diabetic rats exhibited elevated blood glucose, reduced body weight, impaired left ventricular function, increased serum copper levels and myocardial FDX1 expression, decreased L-type calcium currents, and prolonged action potential duration. Quercetin and empagliflozin treatment significantly lowered blood glucose, improved body weight, and restored cardiac function of the diabetic rats, and compared with empagliflozin, quercetin more effectively reduced serum copper levels, downregulated FDX1 expression, and enhanced myocardial L-type calcium currents in diabetic rats. In H9c2 cells, high glucose exposure significantly increased myocardial expressions of FDX1, CK-MB and LDH, which were effectively lowered by quercetin treatment; Elesclomol further elevated FDX1, CK-MB and LDH levels in the exposed cells, and these changes were not significantly affected by the application of quercetin. CONCLUSIONS Quercetin ameliorates myocardial injury in diabetic rats possibly by suppressing myocardial cuproptosis signaling and restoring L-type calcium channel activity.
Animals ; Quercetin/pharmacology* ; Calcium Channels, L-Type/metabolism* ; Diabetes Mellitus, Experimental/metabolism* ; Rats, Sprague-Dawley ; Rats ; Myocytes, Cardiac/drug effects* ; Myocardium/pathology* ; Male

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 prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4 (LDHC4). Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene, and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector. The recombinant vector was introduced into E. coli BL21 (DE3), and LDHC4 protein was obtained by induced expression. The recombinant protein was used as an antigen to immunize New Zealand rabbits, and the antiserum was obtained after three boosted immunizations. The titer of the antiserum against LDHC4 were detected by ELISA. Western blot was used to detect the specificity of the antiserum, and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue. Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51 200. The antibody can be used for Western blot and immunohistochemistry. Conclusion The specific rabbit anti-human LDHC4 polyclonal antibody is successfully prepared.
Humans ; Rabbits ; Animals ; Escherichia coli/genetics* ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; L-Lactate Dehydrogenase/metabolism* ; Blotting, Western ; Antibody Specificity

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*