1.Effect of Serum Containing Zhenwutang on Apoptosis of Myocardial Mast Cells and Mitochondrial Autophagy
Wei TANG ; Meiqun ZHENG ; Xiaolin WANG ; Zhiyong CHEN ; Chi CHE ; Zongqiong LU ; Jiashuai GUO ; Xiaomei ZOU ; Lili XU ; Lin LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):11-21
ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ (AngⅡ) and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment, AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h, and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 (CCK-8) to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of B-type natriuretic peptide (BNP) in cell culture supernatant, and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently, the experiment was divided into a blank group (20% blank serum), a model group (20% blank serum + 5×10-5 mol·L-1 AngⅡ), low-, medium-, and high-dose (5%, 10% and 20%) serum containing Zhenwutang groups, an autophagy inhibitor group (1×10-4 mol·L-1 3-MA), and autophagy inducer group (1×10-7 mol·L-1 rapamycin). The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe (Lyso Tracker) and mitochondrial probe (Mito Tracker) co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect Caspase-3, Caspase-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-related X protein (Bax), and cytochrome C (Cyt C) in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase (Parkin), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3, cleaved Caspase-9, Bax, Bcl-2, and Cyt C in apoptosis-related pathways, phosphorylated ubiquitin ligase (p-Parkin), phosphorylated PTEN-induced kinase 1 (p-PINK1), p62, and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways, and the change of microtubule-associated protein 1 light chain 3 (LC3) Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10-5 mol·L-1, the cell activity was the lowest, and there was no cytotoxicity. At this concentration, the surface area of cardiomyocytes was significantly increased (P<0.01), and the content of BNP in the supernatant of culture medium was significantly increased (P<0.05). Therefore, AngⅡ with a concentration of 5×10-5 mol·L-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group, the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate (P<0.01) and significantly decreased mitochondrial membrane potential (P<0.01). The results of immunofluorescence co-localization showed that compared with the blank group, the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group, the relative mRNA expression of Bax, Caspase-3, Caspase-9, Cyt C, and p62 in the model group was significantly up-regulated (P<0.01), while the relative mRNA expression of Bcl-2, Parkin, and PINK1 was significantly down-regulated (P<0.01). In addition, the relative protein expression of Bax, cleaved Caspase-3, cleaved Caspase-9, Cyt C, and p62 was significantly up-regulated (P<0.01). The LC3Ⅱ/Ⅰ was significantly decreased, and the relative protein expression of Bcl-2, p-Parkin, p-PINK1, and Beclin1 was significantly down-regulated (P<0.01). Compared with the model group, the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate (P<0.01), and the decrease ratio of mitochondrial membrane potential is significantly lowered (P<0.01) in a dose-dependent manner. Additionally, both red and green fluorescence spots became more in these groups. In the 3-MA group, the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax, Caspase-3, Caspase-9, Cyt C, and p62 was significantly down-regulated (P<0.05, P<0.01), while that of Bcl-2, Parkin, and PINK1 was significantly up-regulated (P<0.01). In the serum containing Zhenwutang groups, the relative protein expression levels of Bax, cleaved Caspase-3, cleaved Caspase-9, Cyt C, and p62 were significantly down-regulated (P<0.05,P<0.01). The LC3Ⅱ/Ⅰ was significantly increased, and the relative protein expression levels of Bcl-2, p-Parkin, p-PINK1, and Beclin1 were significantly up-regulated (P<0.01). ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.
2.Clinical practice guidelines for intraoperative cell salvage in patients with malignant tumors
Changtai ZHU ; Ling LI ; Zhiqiang LI ; Xinjian WAN ; Shiyao CHEN ; Jian PAN ; Yi ZHANG ; Xiang REN ; Kun HAN ; Feng ZOU ; Aiqing WEN ; Ruiming RONG ; Rong XIA ; Baohua QIAN ; Xin MA
Chinese Journal of Blood Transfusion 2025;38(2):149-167
Intraoperative cell salvage (IOCS) has been widely applied as an important blood conservation measure in surgical operations. However, there is currently a lack of clinical practice guidelines for the implementation of IOCS in patients with malignant tumors. This report aims to provide clinicians with recommendations on the use of IOCS in patients with malignant tumors based on the review and assessment of the existed evidence. Data were derived from databases such as PubMed, Embase, the Cochrane Library and Wanfang. The guideline development team formulated recommendations based on the quality of evidence, balance of benefits and harms, patient preferences, and health economic assessments. This study constructed seven major clinical questions. The main conclusions of this guideline are as follows: 1) Compared with no perioperative allogeneic blood transfusion (NPABT), perioperative allogeneic blood transfusion (PABT) leads to a more unfavorable prognosis in cancer patients (Recommended); 2) Compared with the transfusion of allogeneic blood or no transfusion, IOCS does not lead to a more unfavorable prognosis in cancer patients (Recommended); 3) The implementation of IOCS in cancer patients is economically feasible (Recommended); 4) Leukocyte depletion filters (LDF) should be used when implementing IOCS in cancer patients (Strongly Recommended); 5) Irradiation treatment of autologous blood to be reinfused can be used when implementing IOCS in cancer patients (Recommended); 6) A careful assessment of the condition of cancer patients (meeting indications and excluding contraindications) should be conducted before implementing IOCS (Strongly Recommended); 7) Informed consent from cancer patients should be obtained when implementing IOCS, with a thorough pre-assessment of the patient's condition and the likelihood of blood loss, adherence to standardized internally audited management procedures, meeting corresponding conditions, and obtaining corresponding qualifications (Recommended). In brief, current evidence indicates that IOCS can be implemented for some malignant tumor patients who need allogeneic blood transfusion after physician full evaluation, and LDF or irradiation should be used during the implementation process.
3.Prognostic correlation analysis of multiple myeloma based on HALP score of peripheral blood before chemotherapy
Min CHEN ; Liying AN ; Xiaojing LIN ; Pan ZHAO ; Xingli ZOU ; Jin WEI ; Xun NI
Chinese Journal of Blood Transfusion 2025;38(1):61-67
[Objective] To explore the predictive value of HALP score for prognosis in patients with multiple myeloma (MM). [Methods] A retrospective analysis was conducted on laboratory indicators and related clinical data of newly diagnosed multiple myeloma (NDMM) patients, treated at the Affiliated Hospital of North Sichuan Medical College from January 2016 to October 2023, prior to their first treatment. The HALP score was calculated, and the optimal cutoff value for HALP was determined using X-tile software. Survival analysis was performed using Kaplan-Meier curves for high HALP and low HALP groups. Univariate and multivariate analyses were conducted using the Cox regression model, and a forest plot was generated using Graphpad Prism to illustrate factors that may impact patient prognosis. The predictive ability of HALP score combined with β2-microglobulin and ECOG score for prognosis in MM patients was evaluated using receiver operating characteristic curve (ROC) analysis. [Results] A total of 203 MM patients were included, with the optimal cutoff value for HALP score being 29.15 (P<0.05). Among them, 101 patients were in the low HALP score group, and 102 patients were in the high HALP score group. The results of univariate and multivariate analysis using the Cox regression model showed that a HALP score <29.15 was an independent risk factor for progression-free survival (PFS) and overall survival (OS) (P<0.05). ROC curve analysis indicated that the combination of HALP score with β2-microglobulin and ECOG score had a higher predictive value for prognosis in MM patients compared to using HALP score alone. [Conclusion] The HALP score is closely related to the prognosis of patients with NDMM. A low HALP score indicates a poorer prognosis, while the combination of HALP score with β2-microglobulin and ECOG score provides a higher predictive value when assessed together.
4.Optimization of temperature parameters for screening unexpected antibodies in Rh system by manual polybrene test
Xin ZOU ; Minjie CHEN ; Sifei MA ; Hongmei YANG
Chinese Journal of Blood Transfusion 2025;38(1):97-100
[Objective] To explore the temperature parameters affecting the polybrene test and determine the optimal temperature conditions for detecting unexpected antibodies of the Rh system. [Methods] The reaction of IgG human anti-D antibody with different dilutions (undiluted, 1∶2, 1∶4, 1∶8, 1∶16, 1∶32,1∶64) with D antigen-positive red blood cells was detected by manual polybrene test (MPT). Different temperatures (25℃ and 37℃) were set, and the reaction time with low ionic medium was 4 minutes. The agglutination integral value of anti-D and red cell depolymerization time were compared to observe the effect of enhanced agglutination reaction, thereby establishing the test temperature reaction conditions for enhancing the MPT. The same reaction condition was applied to 36 blood samples containing unexpected antibodies of the Rh system, and the effect of enhanced MPT was observed in comparison with the polybrene method and the antiglobulin test (column agglutination). [Results] With all other conditions held constant, when low ionic medium was added, the incubation temperature of 25℃ and 37℃ resulted in different total agglutination integral values for anti-D (20.9±2.025 vs 25.5±2.635), and the comparison showed a significant difference (P<0.05). When the antibody dilution was 1∶16, the incubation temperature of 25℃ and 37℃ resulted in different agglutination integral values (3.9±0.738 vs 5.8±0.632), and the comparison showed a significant difference (P<0.05). Erythrocyte depolymerization time (62.8±8.149 vs 90.1±10.713) was significantly different (P<0.05). At a dilution of 1∶32, the incubation temperatures of 25℃ and 37℃ resulted in different agglutination integral values (2.5±0.527 vs 4.3±0.675), as well as different red blood cell dissociation times (35.4±7.792 vs 57.4±10.885)(P<0.05), and the comparison showed a significant difference (P<0.05), with no differences observed in the other groups. In the detection of 36 Rh system unexpected antibody samples, when the antibody titer was ≤2, the enhanced polybrene method had a higher positive rate, and when the antibody titer was ≥4, the detection rates of the three methods were consistent. [Conclusion] The reference temperature condition for the modified MPT is incubation at 37℃ for 4 min after the addition of low ionic medium. The application of this temperature condition to unexpected antibody samples of Rh system could achieve a significant enhancement effect, thereby increasing transfusion safety for the treatment of emergency patients, and is worth popularizing.
5.Current situation and influencing factors of blood pressure measurement cognition and behavior in community patients
Jie YU ; Yawei ZOU ; Xi CHEN ; Junfeng ZHAO ; Yejing WANG
Journal of Public Health and Preventive Medicine 2025;36(1):83-87
Objective To investigate the cognition level and behavior compliance of blood pressure measurement in community residents and analyze the related influencing factors, and to provide evidence for community health management and blood pressure control. Methods A questionnaire survey was conducted to investigate 4470 community patients. Questionnaires included basic personal information,blood pressure measurement cognition, and blood pressure measurement behavior related issues. SPSS 19.0 was used to analyze the basic information, blood pressure measurement cognition, and pressure measurement behavior of the survey subjects. Logistic regression was performed to analyze relevant factors affecting blood pressure measurement cognition and behavior compliance. Results The overall cognitive compliance rate for blood pressure measurement among the visiting community patients was 31.52%. Age, education level, and chronic disease had a statistically significant impact on the cognitive knowledge (P<0.05). The overall behavior compliance rate of blood pressure measurement among the community patients was 23.69%. The cognition, age and education had a statistically significant impact on the overall behavior compliance rate of blood pressure measurement (P<0.05). Conclusion The cognitive level and standardized behavior of blood pressure measurement of community patients need to be improved. More attention should be paid to the elderly, low education level residents and community residents without chronic diseases, to promote community residents to form correct and standardized behavior of blood pressure measurement through health education.
6.Exercise Ameliorates Chronic Restraint Stress-induced Anxiety via PVN CRH Neurons
Jing CHEN ; Cong-Cong CHEN ; Kai-Na ZHANG ; Yu-Lin LAI ; Yang ZOU
Progress in Biochemistry and Biophysics 2025;52(2):501-512
ObjectiveTo investigate the role of paraventricular nucleus (PVN) corticotropin releasing hormone (CRH) neurons in chronic restraint stress (CRS)-induced anxiety-like behavior. And whether exercise relieves chronic restraint stress-induced anxiety through PVN CRH neurons. MethodsTwenty 8-week-old male C57BL/6J mice were randomly divided into control (Ctrl) group and chronic restraint stress (CRS) group. The open field test (OFT) and elevated plus maze (EPM) were used to evaluate anxiety-like behavior of the mice. Food intake was recorded after CRS. Immunofluorescence staining was used to label the expression of c-Fos expression in PVN and calculate the co-expression of c-Fos and CRH neurons. We used chemogenetic activation of PVN CRH neurons to observed the anxiety-like behavior. 8-week treadmill training (10-16 m/min, 60 min/d, 6 d/week) were used to explore the role of exercise in ameliorating CRS-induced anxiety behavior and how PVN CRH neurons involved in it. ResultsCompared with Ctrl group, CRS group exhibited significant anxiety-like behavior. In OFT, the mice in CRS groups spent less time in center area (P<0.001). In EPM, the time in open arm in CRS group were significantly decreased (P<0.001). Besides, food intake was also suppressed in CRS group compared with Ctrl group (P<0.05). Compared with Ctrl group, CRS significantly increase c-Fos expression in PVN and most of CRH neurons co-express c-Fos (P<0.001). Chemogenetic activation of PVN CRH neurons induced anxiety-like behavior (P<0.05) and inhibited feeding behavior (P<0.01). Exercise relieves chronic restraint stress-induced anxiety (P<0.001) and relieved the anorexia caused by chronic restraint stress (P<0.05). Aerobic exercise inhibited the CRS labeled c-Fos in PVN CRH neurons (P<0.001). Furthermore, ablation of PVN CRH neurons attenuated CRS induced anxiety-like behavior. ConclusionCRS activated PVN CRH neurons, induced anxiety-like behavior and reduced food intake. 8-week exercise attenuated CRS-induced anxiety-like behavior through inhibiting PVN CRH neuron. Ablation of CRH PVN neurons ameliorated CRS-induced anxiety-like behavior. These finding reveals a potential neural mechanism of exercise-relieving CRS-induced anxiety-like behavior. This provides a new idea and theoretical basis for the treatment of anxiety and related mental disorders.
7.Effect of Zishen Tongguan Formula on "Gut-prostate" Axis of Rats with Chronic Non-bacterial Prostatitis Based on 16S rDNA Sequencing
Xiran LI ; Mengjiao CHEN ; Kaiping ZOU ; Chenguang ZHAO ; Xingbin DAI ; Xiaoqing ZHANG ; Shun LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):63-71
ObjectiveBased on the theory of "gut-prostate" axis, this study explored the effects and mechanisms of Zishen Tongguan formula and Cinnamomi Cortex in the formula in treating rats with chronic non-bacterial prostatitis(CNP) by detecting the levels of inflammatory factors, and the composition and structure of intestinal flora in CNP rats. MethodsEight out of 42 SD rats were randomly selected as the normal group, and the remaining rats were injected with carrageenan to prepare the CNP model. After successful modeling, 32 rats were randomly divided into the model group, Ningmitai capsule group(0.50 g·kg-1), Zishen Tongguan formula group(2.00 g·kg-1), and the Phellodendri Chinensis Cortex-Anemarrhenae Rhizoma pair group(PCC-AR group, 2.00 g·kg-1), with 8 rats in each group. The administered groups were given the corresponding medicinal solution by gavage, and the normal and model groups were intragastrically administered with an equal volume of normal saline, once a day for 14 consecutive days. The prostate tissues of rats were collected and subjected to hematoxylin-eosin(HE) staining and Masson staining to observe the pathological changes of the tissues in each group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of related inflammatory factors in rat serum, and 16S rDNA sequencing was used to analyze the abundance and diversity changes of gut microbiota before and after administration, and species difference analysis was performed. ResultsAll the administered groups could alleviate the inflammatory symptoms of CNP rats, increase the expression levels of anti-inflammatory factors and decrease the expression levels of pro-inflammatory factors, with the most sIgnificant effect observed in the Zishen Tongguan formula group. Compared with the normal group, the expression levels of interleukin(IL)-8, hypersensitive C-reactive protein(hs-CRP), immunoglobulin(Ig)M, secretory IgA (sIgA), and inducible nitric oxide synthase(iNOS) were sIgnificantly increased in the model group(P<0.01). Compared with the model group, the expression levels of the above inflammatory factors in all administered groups were significantly reduced(P<0.01). When compared with the PCC-AR group, the Zishen Tongguan formula group showed a significant decrease in transforming growth factor(TGF)-β1 expression level(P<0.05) and a significant increase in IgM expression level(P<0.01). The results of gut microbiota analysis showed that, compared with the PCC-AR group, at the order level, the Zishen Tongguan formula group significantly reduced the relative abundance of conditional pathogens such as Bacteroidales, Acidaminococcales, Rhodospirillales, Clostridiales, and Elusimicrobiales(P<0.01). And at the genus level, the Zishen Tongguan formula group significantly decreased the relative abundance of pathogenic microbiota such as Lachnospira and Bacteroides(P<0.01) and significantly increased the relative abundances of beneficial microbiota such as Ruminococcus and Lactobacillus(P<0.01). ConclusionZishen Tongguan formula can reduce the level of harmful intestinal bacteria, increase the level of beneficial intestinal bacteria, down-regulate the expression of serum inflammatory factors, and the small amount of Cinnamomi Cortex in the formula may play a key role in the treatment of CNP with this formula.
8.Effect of Zishen Tongguan Formula on "Gut-prostate" Axis of Rats with Chronic Non-bacterial Prostatitis Based on 16S rDNA Sequencing
Xiran LI ; Mengjiao CHEN ; Kaiping ZOU ; Chenguang ZHAO ; Xingbin DAI ; Xiaoqing ZHANG ; Shun LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):63-71
ObjectiveBased on the theory of "gut-prostate" axis, this study explored the effects and mechanisms of Zishen Tongguan formula and Cinnamomi Cortex in the formula in treating rats with chronic non-bacterial prostatitis(CNP) by detecting the levels of inflammatory factors, and the composition and structure of intestinal flora in CNP rats. MethodsEight out of 42 SD rats were randomly selected as the normal group, and the remaining rats were injected with carrageenan to prepare the CNP model. After successful modeling, 32 rats were randomly divided into the model group, Ningmitai capsule group(0.50 g·kg-1), Zishen Tongguan formula group(2.00 g·kg-1), and the Phellodendri Chinensis Cortex-Anemarrhenae Rhizoma pair group(PCC-AR group, 2.00 g·kg-1), with 8 rats in each group. The administered groups were given the corresponding medicinal solution by gavage, and the normal and model groups were intragastrically administered with an equal volume of normal saline, once a day for 14 consecutive days. The prostate tissues of rats were collected and subjected to hematoxylin-eosin(HE) staining and Masson staining to observe the pathological changes of the tissues in each group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of related inflammatory factors in rat serum, and 16S rDNA sequencing was used to analyze the abundance and diversity changes of gut microbiota before and after administration, and species difference analysis was performed. ResultsAll the administered groups could alleviate the inflammatory symptoms of CNP rats, increase the expression levels of anti-inflammatory factors and decrease the expression levels of pro-inflammatory factors, with the most sIgnificant effect observed in the Zishen Tongguan formula group. Compared with the normal group, the expression levels of interleukin(IL)-8, hypersensitive C-reactive protein(hs-CRP), immunoglobulin(Ig)M, secretory IgA (sIgA), and inducible nitric oxide synthase(iNOS) were sIgnificantly increased in the model group(P<0.01). Compared with the model group, the expression levels of the above inflammatory factors in all administered groups were significantly reduced(P<0.01). When compared with the PCC-AR group, the Zishen Tongguan formula group showed a significant decrease in transforming growth factor(TGF)-β1 expression level(P<0.05) and a significant increase in IgM expression level(P<0.01). The results of gut microbiota analysis showed that, compared with the PCC-AR group, at the order level, the Zishen Tongguan formula group significantly reduced the relative abundance of conditional pathogens such as Bacteroidales, Acidaminococcales, Rhodospirillales, Clostridiales, and Elusimicrobiales(P<0.01). And at the genus level, the Zishen Tongguan formula group significantly decreased the relative abundance of pathogenic microbiota such as Lachnospira and Bacteroides(P<0.01) and significantly increased the relative abundances of beneficial microbiota such as Ruminococcus and Lactobacillus(P<0.01). ConclusionZishen Tongguan formula can reduce the level of harmful intestinal bacteria, increase the level of beneficial intestinal bacteria, down-regulate the expression of serum inflammatory factors, and the small amount of Cinnamomi Cortex in the formula may play a key role in the treatment of CNP with this formula.
9.Depressive symptoms and associated factors among middle school and college students from 2021 to 2023 in Hunan Province
Chinese Journal of School Health 2025;46(1):96-101
Objective:
To investigate the current status and trends of depressive symptoms among middle school and college students in Hunan Province, and to explore the primary related factors of depressive symptoms, so as to provide a scientific basis for strengthening mental health among students.
Methods:
A total of 279 382 students in Hunan Province were selected through a stratified cluster random sampling method from 2021 to 2023. National Survey Questionnaire on Common Diseases and Health Influencing Factors among Students was adopted for the survey, and the Center for Epidemiological Studies Depression Scale was used to assess their depressive symptoms. The χ 2 test and trend χ 2 test were used to analyze depressive symptoms prevalence and trends, and multivariable Logistic regression was used to analyze the related factors of depressive symptoms.
Results:
The prevalence of depressive symptoms among students in Hunan Province from 2021 to 2023 were 19.66%, 20.17% and 21.47%, respectively, showing an upward trend ( χ 2 trend =9.07, P <0.01). In addition, the results of the multivariable Logistic regression analysis showed that students with healthy diet ( OR=0.43, 95%CI =0.40-0.45), adequate sleep ( OR=0.88, 95%CI =0.86-0.90), and acceptable screen time ( OR=0.61, 95%CI =0.60-0.62) had lower risks in depressive symptoms detection, while students with smoking ( OR= 1.95, 95%CI =1.88-2.02), secondhand smoke exposure ( OR=1.33, 95%CI =1.30-1.36) and Internet addiction ( OR= 4.19 , 95%CI =4.05-4.34) had higher risks in depressive symptoms detection, with differences in the degree of association among different genders, educational stages and urban rural groups ( OR=0.40-6.04, Z =-12.69-11.98) ( P <0.05).
Conclusions
There is an increasing trend of depressive symptoms among middle school and college students in Hunan Province from 2021 to 2023.Targeted depression prevention measures should be taken for students with different demographic characteristics to promote their mental health.
10.MiRSNP in MGMT 3′ untranslated region regulates low-dose radiation-induced senescence in lung cells
Lingyu ZHANG ; Yashi CAI ; Huixian LI ; Min ZHANG ; Changyong WEN ; Weixu HUANG ; Huifeng CHEN ; Jianming ZOU
China Occupational Medicine 2025;52(1):25-32
Objective To investigate the role of the O-6-methylguanine-DNA methyltransferase (MGMT) gene-3′ untranslated region (UTR) microRNA-associated single nucleotide polymorphism (miRSNP) (rs7896488 G>A) in affecting miR-4297-targeted modulation of MGMT in senescence of lung cells with polymorphic genotypes induced by fractionated low dose ionizing radiation (LDIR). Methods i) MiRSNPs were predicted and screened using bioinformatics, and DNA from two types of lung cells, A549 cells and human bronchial epithelioid cells (HBE cells), was extracted for target gene sequencing. After co-transfection of pGL3c-MGMT-3′UTR-rs7896488 G>A reporter gene recombinant plasmid, pRL-TK Vector with micrON mimic NC #22 or micrON hsa-miR-4297 mimic (set up as the mimic NC group and the miR-4297 mimic group) in these two types of lung cells, dual luciferase reporter gene assay was performed. The relative expression of MGMT mRNA was detected by real-time fluorescence quantitative polymerase chain reaction, and the relative expression of MGMT protein was detected by Western blotting. ii) These two types of lung cells were randomly divided into the control group and irradiation group, which received either 0 or 100 mGy X-rays irradiation seven times. After irradiation, the cells were transfected with either micrON mimic NC #22 or micrON hsa-miR-4297 mimic, resulting in mimic NC + control group, miR-4297 mimic + control group, mimic NC + irradiation group, and miR-4297 mimic + irradiation group. Cells were collected for senescence-associated-β-galactosidase (SA-β-Gal) staining, and the relative expression of matrix metalloproteinase-9 (MMP-9) and chemokine (C-X-C motif) ligand-1 (CXCL-1) proteins was detected via Western blotting. Results i) The rs7896488 G>A was the miRSNP located in the conserved binding region targeted by miR-4297 in the MGMT gene 3′UTR. A549 cells were the rs7896488 GG wild-type homozygous genotype, while HBE cells were the rs7896488 GA heterozygous mutant genotype. In the miR-4297 mimic group, A549 and HBE cells carrying the rs7896488 G allele showed significantly lower dual-luciferase activity compared with that in the mimic NC group (both P<0.01). However, there was no significant difference in dual-luciferase activity between the two groups in both A549 and HBE cells carrying the rs7896488 A allele (both P>0.05). The relative expression levels of MGMT mRNA and MGMT protein of A549 cells in the miR-4297 mimic group were lower than those in the mimic NC group (both P<0.05). However, there was no significant difference in MGMT mRNA and MGMT protein of HBE cells between these two groups (both P>0.05). ii) The relative activity of SA-β-Gal and the relative expression of MMP-9 and CXCL-1 proteins of A549 cells in the miR-4297 mimic+irradiation group were higher than those in the mimic NC + control group, the miR-4297 mimic + control group, and the mimic NC + irradiation group (all P<0.05). The relative activity of SA-β-Gal and the relative expression of MMP-9 and CXCL-1 proteins of HBE cells in the miR-4297 mimic + irradiation group were higher than those in the mimic NC + control group and the miR-4297 mimic + control group (all P<0.05), while there was no significant difference compared with those in the mimic NC + irradiation group (all P>0.05). Conclusion MGMT-3′UTR-miRSNP rs7896488 G>A plays a role in LDIR-induced senescence of lung cells with different polymorphic genotypes by affecting miR-4297-targeted regulation of MGMT.


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