The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.
Animals ; Rats ; PC12 Cells ; Ferroptosis/genetics* ; Reactive Oxygen Species ; Transcription Factors ; Glutathione

Squalene epoxidase (SQLE) is a potential target for the treatment of liver cancer. Bioinformatics analysis indicated that the high expression of SQLE was closely related to the clinical stage and poor prognosis of patients with liver cancer. However, the existing inhibitors against SQLE 195 tyrosine residue (Y195) cannot be used clinically due to severe side effects. In this study, 35 small-molecule compounds targeting SQLE 335 tyrosine residue (Y335) were selected by computer virtual screening. Combined with MTT assay, 3 candidate compounds (19^#, 31^# and 35^#) with significant inhibitory effects on the proliferation of Huh7 cell line were obtained. Further studies showed that these 3 compounds could inhibit the migration of Huh7 cells, reduce the contents of total and free cholesterol, up-regulate the expression of tumor suppressor gene PTEN, and down-regulate the expression of PI3K and AKT proteins. The results showed that the novel inhibitors 19^#, 31^# and 35^# targeting SQLE Y335 could reduce cholesterol content, inhibit the proliferation and migration of Huh7, thus playing an anti-liver cancer role.

OBJECTIVE: To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. RESULTS: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. CONCLUSION The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.
Humans ; Carcinoma, Renal Cell/pathology* ; Cell Proliferation ; Hypoxia ; Kidney Neoplasms ; MicroRNAs/genetics* ; Neoplasm Invasiveness/genetics* ; RNA, Circular/metabolism* ; RNA, Small Interfering ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics*

OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC₅₀) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC₅₀ were significantly higher than those of the sensitive cell lines (10.99 μmol/L, P < 0.01; 11.96 μmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 μmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.
Humans ; Carcinoma, Renal Cell/metabolism* ; Axitinib/pharmacology* ; Kidney Neoplasms/metabolism* ; bcl-2-Associated X Protein ; Nuclear Proteins ; Cell Line, Tumor ; Apoptosis ; Cell Proliferation

BACKGROUND: The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults. METHODS: Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 μg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 μg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose. RESULTS: In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-μg vaccine (n = 24), 10-μg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-μg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-μg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-μg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-μg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses. CONCLUSIONS: Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-μg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial. TRIAL REGISTRATION http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).
Adult ; COVID-19 ; COVID-19 Vaccines ; Double-Blind Method ; Humans ; SARS-CoV-2 ; Vaccines, Inactivated/adverse effects*

Plants have a memory function for the environmental stress they have suffered. When they are subjected to repeated environmental stress, they can quickly and better activate the response and adaptation mechanism to environmental stress, thus realizing long-term stable reproduction. However, most of the relevant studies are applied to crops and Arabidopsis thaliana rather than medicinal plants about the improvement of plant growth status and the effect on phytoalexin biosynthesis. In this study, yeast extract(YE) was used as an elicitor to simulate biotic stress, and the changes in biomass and the content of some secondary metabolites were measured by giving repeated stresses to Sorbus aucuparia suspension cell(SASC). The results showed that the accumulation levels of biomass and some secondary metabolites in SASC subjected to repeated stress are significantly increased at some time points compared with single stress. A phenomenon that SASC can memorize biotic stress is confirmed in this study and influences phytoalexin accumulation in SASC. Furthermore, the work laid the groundwork for research into the transgenerational stress memory mechanism of medicinal plant.
Cells, Cultured ; Secondary Metabolism ; Sorbus ; Stress, Physiological

Objective: To obtain the glycosyltransferase gene involved in modification reaction of phytoalexin from Sorbus pohuashanensis suspension cell,and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data,specific primers were designed to obtain 2 cDNA sequences of SaUGTs genes,construct prokaryotic expression vector HIS-MBP-pET28a-SaUGTs and induce the expression of recombinant SaUGTs protein. Result: SaUGT1 and SaUGT2 sequences were cloned and obtained from glycosyltransferases,then bioinformatic analysis of the sequence and prokaryotic expression analysis were conducted. SaUGT1 gene contained 1 458 bp open reading frame (ORF),encoding a polypeptide of 485 amino acids,with a relative molecular weight of 54.27 kDa and theoretical isoelectric point (pI) of 5.50.SaUGT2 gene contained 1 431 bp ORF,encoding a polypeptide of 476 amino acids,with a relative molecular weight of 53.49 kDa and theoretical pI of 5.63. Bioinformatics analysis indicated that SaUGT1 and SaUGT2 protein had no signal peptide,and the conserved domains of glycosyltransferase family were detected. Phylogenetic results showed that SaUGT1 and SaUGT2 proteins had the closest relationship with the UGT85 family of A. thaliana. Differential expression analysis revealed that the relative expression levels of SaUGT1 and SaUGT2 were increased significantly after being induced by yeast extract (YE), with the highest expression level found at 24 h and 12 h. The recombinant SaUGT1 and SaUGT2 proteins were successfully expressed in Escherichia coli DE3 cells and finally,the recombinant SaUGT1 and SaUGT2 proteins were purified through Ni²⁺ affinity chromatography. Conclusion: The glycosyltransferase gene was cloned from the S. aucuparia for the first time,and the prokaryotic expression vector was successfully constructed,laying foundation for further study of the function of this gene.

OBJECTIVE: To estimate the detrimental effects of shortwave exposure on rat hippocampal structure and function and explore the underlying mechanisms. METHODS: One hundred Wistar rats were randomly divided into four groups (25 rats per group) and exposed to 27 MHz continuous shortwave at a power density of 5, 10, or 30 mW/cm2 for 6 min once only or underwent sham exposure for the control. The spatial learning and memory, electroencephalogram (EEG), hippocampal structure and Nissl bodies were analysed. Furthermore, the expressions of N-methyl-D-aspartate receptor (NMDAR) subunits (NR1, NR2A, and NR2B), cAMP responsive element-binding protein (CREB) and phosphorylated CREB (p-CREB) in hippocampal tissue were analysed on 1, 7, and 14 days after exposure. RESULTS: The rats in the 10 and 30 mW/cm2 groups had poor learning and memory, disrupted EEG oscillations, and injured hippocampal structures, including hippocampal neurons degeneration, mitochondria cavitation and blood capillaries swelling. The Nissl body content was also reduced in the exposure groups. Moreover, the hippocampal tissue in the 30 mW/cm2 group had increased expressions of NR2A and NR2B and decreased levels of CREB and p-CREB. CONCLUSION Shortwave exposure (27 MHz, with an average power density of 10 and 30 mW/cm2) impaired rats' spatial learning and memory and caused a series of dose-dependent pathophysiological changes. Moreover, NMDAR-related CREB pathway suppression might be involved in shortwave-induced structural and functional impairments in the rat hippocampus.
Animals ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Dose-Response Relationship, Radiation ; Electroencephalography ; radiation effects ; Hippocampus ; radiation effects ; Male ; Memory ; radiation effects ; Nissl Bodies ; physiology ; radiation effects ; Radio Waves ; adverse effects ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; genetics ; metabolism ; Spatial Learning ; radiation effects

OBJECTIVE: To explore long-term following-up clinical effects of lateral closed high tibial osteotomy for the treatment of knee osteoarthritis. METHODS: Twenty patients with medial unicompartmental knee osteoarthritis were treated with lateral closed high tibial osteotomy and screw fixation from June 2005 to December 2015. Among them, including 17 females and 3 males, aged from 43 to 76 years old with an average of (57.80±8.05) years old. VAS score and KSS score were applied to evaluate recovery degree of pain and function before operation and after operation at 1, 5 and 10 years, and postoperative complications were observed. RESULTS: Sixteen patients were followed-up, the time ranged from 9 to 11(10.0±0.8) years, 4 patients were loss to follow-up. Preoperative VAS score was 7.88±1.15 and decreased to 3.19±0.91, 3.44±0.96, 3.69±1.20 at 1, 5 and 10 years after operation, and there were statistical differences in VAS score between before and after operation at different time points (<0.05). Clinical score of KSS increased from 61.94±5.74 before opertaion to 75.50±4.62, 80.13±3.97, 77.38±6.40 at 1, 5 and 10 years after operation, and there were statistical differences in clinical score of KSS between before and after operation at different time points(<0.05); functional score of KSS increased from 62.81±13.03 before operation to 77.50±8.56, 81.88±6.55, 76.88±10.78, and there were statistical differences in functional score of KSS between before and after operation at different time points(<0.05). All incisions healed well without complications such as fibula nerve injury and fracture nonunion. CONCLUSIONS Lateral closed high tibial osteotomy and screw fixation for knee osteoarthritis could receive good clinical results, stop and delay progress of knee osteoarthritis, and long-term following-up could achieve the same effect as total knee arthroplasty.
Adult ; Aged ; Female ; Follow-Up Studies ; Humans ; Knee Joint ; Male ; Middle Aged ; Osteoarthritis, Knee ; surgery ; Osteotomy ; Tibia ; Treatment Outcome

The purpose of this study was to explore the differences in interhemispheric functional connectivity in patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (aMCI) based on a triple network model consisting of the default mode network (DMN), salience network (SN), and executive control network (ECN). The technique of voxel-mirrored homotopic connectivity (VMHC) analysis was applied to explore the aberrant connectivity of all patients. The results showed that: (1) the statistically significant connections of interhemispheric brain regions included DMN-related brain regions (i.e. precuneus, calcarine, fusiform, cuneus, lingual gyrus, temporal inferior gyrus, and hippocampus), SN-related brain regions (i.e. frontoinsular cortex), and ECN-related brain regions (i.e. frontal middle gyrus and frontal inferior); (2) the precuneus and frontal middle gyrus in the AD group exhibited lower VMHC values than those in the aMCI and healthy control (HC) groups, but no significant difference was observed between the aMCI and HC groups; and (3) significant correlations were found between peak VMHC results from the precuneus and Mini Mental State Examination (MMSE) and Montreal Cognitive Scale (MOCA) scores and their factor scores in the AD, aMCI, and AD plus aMCI groups, and between the results from the frontal middle gyrus and MOCA factor scores in the aMCI group. These findings indicated that impaired interhemispheric functional connectivity was observed in AD and could be a sensitive neuroimaging biomarker for AD. More specifically, the DMN was inhibited, while the SN and ECN were excited. VMHC results were correlated with MMSE and MOCA scores, highlighting that VMHC could be a sensitive neuroimaging biomarker for AD and the progression from aMCI to AD.
Aged ; Aged, 80 and over ; Alzheimer Disease/physiopathology* ; Brain/diagnostic imaging* ; Brain Mapping ; Cognitive Dysfunction/physiopathology* ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Memory ; Middle Aged ; Models, Neurological ; Nerve Net