OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Objective:To evaluate the efficacy and safety of antaitasvir phosphate 100 mg or 200 mg combined with yiqibuvir for 12 weeks in patients with various genotypes of chronic hepatitis C, without cirrhosis or compensated stage cirrhosis.Methods:Patients with chronic hepatitis C (without cirrhosis or compensated stage cirrhosis) were randomly assigned to the antaitasvir phosphate 100 mg+yiqibuvir 600 mg group (100 mg group) or the antaitasvir phosphate 200 mg+yiqibuvir 600 mg group (200 mg group) in a 1∶1 ratio. The drugs were continuously administered once a day for 12 weeks and observed for 24 weeks after drug withdrawal. The drug safety profile was assessed concurrently with the observation of the sustained virological response (SVR12) in the two patient groups 12 weeks following the drug cessation. The intention-to-treat concept was used to define as closely as possible a full analysis set, including all randomized cases who received the experimental drug at least once. The safety set was collected from all subjects who received the experimental drug at least once (regardless of whether they participated in the randomization group) in this study. All efficacy endpoints and safety profile data were summarized using descriptive statistics. The primary efficacy endpoint was SVR12. The primary analysis was performed on a full analysis set. The frequency and proportion of cases were calculated in the experimental drug group (antaitasvir phosphate capsules combined with yiqibuvir tablets) that achieved "HCV RNA

Objective:To study the differences of intestinal flora between neonatal rats with intrauterine hypoxia and healthy neonatal rats using high-throughput sequencing technology to determine the effects of intrauterine hypoxia on neonatal intestinal flora.Methods:Intrauterine hypoxia model were established in neonatal rats. On d1 and d7 after birth, intestinal samples were collected from intrauterine hypoxic group and normal control group and assigned into INH1 group (intrauterine hypoxia d1), INH7 group (intrauterine hypoxia d7), NOR1 group (normal control d1) and NOR7 group (normal control d7). 16S rRNA sequencing were conducted using these samples and the differences in the diversity, richness and composition of the flora among the groups were compared.Results:(1) The Alpha diversity of the intestinal flora in the INH1 group was higher than the NOR1 group. Specifically, both sobs and chao indices, representing the richness of the flora, in INH1 group were significantly higher than the NOR1 group (sobs index: 114.5±35.6 vs. 50.5±21.3, chao index: 135.6±38.5 vs. 73.9±28.8)( P<0.05). Compared with the NOR7 group, the mean values of sobs, ace, chao, simpson and shannon indices in the INH7 group showed no significant differences ( P>0.05). (2) At the phylum and genus level, the dominant bacterial groups in the intrauterine hypoxia group on d1 were firmicutes and streptococcus and proteus and escherichia for the normal control group. The difference of intestinal flora between intrauterine hypoxia group and the normal control group on d7 was smaller than the difference between the two groups on d1. Compared with INH1 group, the INH7 group had increased escherichia composition and decreased streptococcus composition. Conclusions:Intrauterine hypoxia changes the initial colonization and later affects the abundance and structural composition of the intestinal flora in newborn rats.

Objective: To investigate nutritional quality of school lunch in some primary schools and middle schools in the Pearl River Delta, and to provide the scientific basis for improving the nutritional quality of students lunch and formulating scientific and effective interventions. Methods: Five-day lunch meal survey by chemical analysis were conducted, and students lunch at school were recorded by meal review in three age groups from 8 primary and middle schools in the Pear River Delat area. The energy and nutrient content were obtained and compared with the reference intake of dietary nutrients of student. Results: The average protein intake at lunch of all age groups had reached the recommended standard (80%-95%), the energy supply ratio of carbohydrate in the range of 38.3%-42.3%, the energy supply ratio of fat in 63% school meal exceeded the recommended standard. Vitamin A, vitamin B 1, vitamin B 2, calcium, iron and other nutrients were seriously inadequate; while sodium intake far exceeded the recommended standard. Conclusion The main nutrients of school lunch of primary and middle school in Pearl River Delta can basically meet the growth and development needs, but there are still some deficiency and unbalanced diet nutrient content which are lower than the recommended intake. It is recommended to strengthen nutrition education of catering enterprises and school to improve the scientific combination of diets.

Objective To investigate the 18F-FDG PET/CT imaging features of thoracolumbar tuberculosis.Methods Nine-ty-eight patients with thoracolumbar vertebral column destruction receiving 18F-FDG PET/CT examination in our hospital from May 2005 to January 2017 were selected as the study subjects and divided into the tuberculosis(TB) group (n=27) and non-TB group (n=71) according to the result of pathology examination and follow-up.The 18F-FDG PET/CT signs were compared between the two groups,and the binary classification Logistic regression was used to screen out the signs with statistical significance.Results Forty-two lesions and 114 lesions were detected in the TB group and non-TB group respectively.The incidence rates of continuous vertebral body involvement,intervertebral disc lesion,vertebral body compression fractures,cold abscess," radioactive cold area",high lesion SUVmax and slightly higher SUVmax sign in the TB group were higher than those in the non-TB group(P＜ 0.05) The binary classification Logistic regression analysis results showed that continuous vertebral involvement,intervertebral disc lesion,vertebral body compression fractures,cold abscess and "radioactive cold area" were the independent factors for diagnosis of thoracolumbar TB(P＜0.05).The sensitivity,specificity,positive prediction value,negative predictionvalue and Youden index in the combination of continuous vertebral involvement + intervertebral disc lesion were 71.4％,81.6％,58.8％,88.6％,53.0％,respectively.Conclusion For the diagnosis of thoracolumbar TB,the combination of continuous vertebral involvement + intervertebral disc lesion has the highest diagnostic value.

Objective To perform the performance verification of the Sysmex XT-2000i hematology analyzer.Methods Accord-ing to requirements of NCCLS EP5-A2,EP15-A2,EP6-A2,EP9-A2 documents,the accuracy,precision and linearity of the Symex XT-2000i hematology were evaluated and the detection results were compared with those detected by the Sysmex XE-2100i which participated in the external quality assessment(EQA)of Ministry of Public Health for long time.Results The accuracy,precision, linearity and contamination rate all are within the permissible range.Conclusion The performance of Sysmex XT-2000i is good,the determination results are reliable.As a perfect hematology analyzer,it can satisfy the hematological analysis of hospital clinical la-boratory.

It is kno wn that 8-Br-cAMP is one of selective bi nding site analogues for cAMP RIIα to af fect cell growth through regulation of g ene expression.The p16,p21wafl,p53 a nd Rb are antioncogenes which affect cel l growth through control of cell cycle.T he aim of this study is to investigate t he 8-Br-cAMP effect on the expression of antioncogenes in human HXO-Rb44 cells. Methods Cultured HXO-Rb44 cells in RPMI -1 640 medium were divided into two aliquot s.8-Br-cAMP (2×10-5mol/L) was added i nto one aliquot for 24h as the experime ntal group(EG),the another aliquot witho ut 8-Br-cAMP as the control group(CG).Af ter 24h,the cell suspension was dropped onto the nitrocellulose membrane.The mR NA of p16,p21wafl,wild type(w)p53,mut ant type(m) p53 and Rb were used respec tively with biotin-labeled cDNA probes b y intact cell RNA dot blot.The immunorea ctivity(IR) of P16,P21wafl,PRb,PCN A,cdk2 and cdk4 were detected respecti vely with specific monoclonal antibodies on dot blot.ResultsThe mRNA dot blot s ignals of mp53 and protein dot blot of cdk2-IR,cdk4-IR and PCNA-IR in EG were weaker than those in CG(P＜0.05～0.01). W hile,the mRNA signals of p16,p21wafl,wp53 and Rb in EG were stronger than tho se in CG(P＜0.05～0.01).The intensity of ea ch protein dot blot was consistent with that of their RNA dot blot (except for w P53-IR and mP53-IR not to be done).Conc lusions(1)8-Br-cAMP could up-regul ate expression of antioncogenes includin g p16,p21wafl,wp53,Rb,and protein exp ression of P16,P21wafl and PRb.(2) 8-Br-cAMP could down-regulate mp53 gene expression and protein expression of cd k2,cdk4 and PCNA.The results suggest t hat 8-Br-cAMP could inhibit human HXO-Rb 44 cell growth through interfering rela ted gene expression of cell cycle.