BACKGROUND:Intervertebral disc degeneration is clinically considered to be the main cause of low back pain,but due to the unclear pathogenesis of intervertebral disc degeneration,there is still a lack of effective means to delay the progression of the disease.Single-cell RNA sequencing technology can amplify and sequence mRNA at the single-cell level,reveal the gene expression intensity of a single cell,discover different cell subsets in tissues according to the heterogeneity of cells,study the pathogenesis of intervertebral disc degeneration at the molecular level,and provide a new theoretical basis for its early diagnosis and treatment. OBJECTIVE:To introduce the basic principles of single-cell RNA sequencing technology and review the research progress of single-cell RNA sequencing technology in intervertebral disc degeneration in recent years. METHODS:A computer was used to search PubMed,Web of Science,CNKI and WanFang databases for the literature published from 2012 to 2022.Key words were"single-cell RNA sequencing,intervertebral disc degeneration,sequencing Technology"in Chinese and English.Duplicate,poor-quality and irrelevant articles were excluded;a total of 70 articles were eventually included. RESULTS AND CONCLUSION:(1)We identified new cell subsets such as homeostatic chondrocytes,hypertrophy chondrocyte-like nucleus pulposus cells and fibrous nucleus pulposus cells,identified the marker genes and transcription factors of these cell subsets,and described the functions,differentiation paths and cell fate of these cell subsets during the development and progression of intervertebral disc degeneration,and proposed the concept of progenitor nucleus pulposus cells.A cell subpopulation with progenitor nucleus pulposus cells properties was identified and its effectiveness in treating intervertebral disc degeneration was verified in mice.(2)Fibro chondrocyte-like annulus fibrosus cells and annulus fibrosus stem cells with both cartilage and fiber properties were identified,and a new type of composite hydrogel was prepared by combining fibrous cartilage inducers silk fibroin and hyaluronic acid in vitro.Experiments in mice demonstrated that this hydrogel could repair both annulus fibrosus tissue and cartilage matrix,and was remarkably effective in the treatment of intervertebral disc degeneration.(3)Regulatory chondrocytes were found in endplate cartilage.Two distinct fates in the progression of intervertebral disc degeneration were analyzed and the differential genes in the two fates were identified.Intercellular communication analysis indicated that regulatory chondrocytes interact with endothelial cells to promote angiogenesis.(4)Immune cells such as macrophages,T cells,myeloid progenitor cells and neutrophils were identified in the degenerated intervertebral disc tissues,demonstrating the existence of immune response during intervertebral disc degeneration.It was found that apolipoprotein induced the polarization of macrophages M1 and M2 subtypes,and this polarization process affected the activity of progenitor nucleus pulposus cells by amplifying the inflammatory response through the MIF signaling pathway.

Objective:To evaluate the effect of tubastatin A (TubA) on pyroptosis during brain injury after cardiac arrest and resuscitation in swine.Methods:Twenty-two conventional male white swine, weighing 34-39 kg, aged 4-6 months, were divided into 3 groups using a random number table: sham operation group (group S, n=6), cardiac arrest-cardiopulmonary resuscitation (CA-CPR) group ( n=8) and CA-CPR+ TubA group ( n=8). The swine model of CA-CPR was established by 9 min of cardiac arrest and 6 min of cardiopulmonary resuscitation in CA-CPR group and CA-CPR+ TubA group. TubA 4.5 mg/kg (in 50 ml of normal saline) was infused over 1 h via the femoral vein starting from 5 min after resuscitation in CA-CPR+ TubA group. Before developing the model and at 1, 2, 4 and 24 h after resuscitation (T 0-4), blood samples were collected from the femoral vein for determination of the concentrations of neuron specific enolase (NSE) and S100β protein in serum (by enzyme-linked immunosorbent assay). Neurological deficit score (NDS) was evaluated at T 4. The animals were then sacrificed, and their brain cortex tissues were harvested to measure the expression of histone deacetylase 6 (HDAC6), caspase-3, cleaved caspase-3, gasdermin E (GSDME) and GSDME N-terminal (N-GSDME) (by Western blot) and contents of high mobility group box 1 (HMGB1), interleukin-1β (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the serum concentrations of NSE and S100β were significantly increased at T 1-4, NDS was increased at T 4, the expression of HDAC6, caspase-3, cleaved caspase-3, GSDME and N-GSDME in brain cortex was up-regulated, and the contents of HMGB1, IL-1β and IL-18 were increased in CA-CPR and CA-CPR+ TubA groups ( P<0.05). Compared with group CA-CPR, the serum concentrations of NSE and S100β were significantly decreased at T 3, 4, NDS was decreased at T 4, the expression of HDAC6, caspase-3, cleaved caspase-3, GSDME and N-GSDME in brain cortex was down-regulated, and the contents of HMGB1, IL-1β and IL-18 were decreased in group CA-CPR+ TubA ( P<0.05). Conclusions:The mechanism by which TubA alleviates brain injury after cardiac arrest and resuscitation may be related to inhibition of pyroptosis in swine.

Objective:To study the effects and the related molecular mechanisms of miR-148a-3p on the proliferation,invasion and migration of salivary adenoid cystic carcinoma SACC-LM cells.Methods:miR-148a-3p mimics and inhibitors,siRNA targeting EG-FR and their corresponding controls were transfected into SACC-LM cells.Bioinformatics was used to predict the potential target genes of miR-148a-3p.EGFR and miR-148a-3p mRNA expression levels were examined by qRT-PCR and the protein levels of EG-FR were detected by Western blotting.CCK-8,scratch,and Transwell assays were used to study the proliferation,migration,and invasion of SACC-LM cells,respectively.The direct targeting relationship between miR-148a-3p and EGFR was examined by using the double luciferase reporter gene assay.Statistical analysis of the data was performed by SPSS 22.0 software.Results:Overexpres-sion or inhibition of miR-148a-3p significantly inhibited or promoted the proliferation,invasion and metastasis of SACC-LM cells re-spectively(P＜0.05).Bioinformatics and double luciferase assay showed that miR-148a-3p directly targeted and regulated the expres-sion of EGFR(P＜0.001).Downregulation of EGFR inhibited the proliferation,migration and invasion of SACC-LM cells(P＜0.05)and partially reversed the promoting effect of miR-148a-3p inhibition(P＜0.05).Conclusion:The downregulation of miR-148a-3p leads to the abnormally high expression of its target gene EGFR,and promotes the proliferation,invasion,and migration of salivary adenoid cystic carcinoma cells.

Objective:To investigate the effects of methyltransferases like 3(METTL3)mediated m6A modification of double homology cassette A pseudogene8(DUXAP8)on the proliferation,migration and invasion of salivary adenoid cystic carcinoma SACC-LM cells and its potential molecular mechanisms.Methods:Whole-transcriptome sequencing showed that DUXAP8 was highly ex-pressed in SACC than in para-cancerous tissues(P＜0.05).The m6A modification sites on DUXAP8 were predicted using the SRAMP website,and the mRNA and protein expression of m6A-modified genes and the genes associated with the epithelial-mesen-chymal transition(EMT)was measured by qRT-PCR and Western blot,respectively.METTL3 and DUXAP8 was knocked down or overexpressed in SACC-LM cells,and the proliferation,migration,and invasion of the cells were assessed by CCK-8,scratch and Transwell assays.The correlation between METTL3 and DUXAP8 was evaluated using MeRIP-qPCR.Results:The expression of DUXAP8 in SACC tumor was higher than that in para-cancerous tissues(P＜0.05).Knockdown of DUXAP8 reduced proliferation,migration and invasion of SACC-LM cells,as well as the expression of EMT-related genes(P＜0.05).Multiple m6A modification sites of high confidence were found on DUXAP8.METTL3 was highly expressed in tumor tissues,more than other related genes(P＜0.05)and enzyme-encoding genes in SACC-LM cells(P＜0.05).METTL3 was found to function as a methyltransferase to regulate the expression of DUX-AP8,and downregulation of METTL3 inhibited prolifera-tion,migration and invasion of SACC-LM cells and partially reversed the promotion of these activities induced by DUX-AP8 overexpression(P＜0.05).Conclusion:METTL3-me-diated m6A modification upregulated DUXAP8 expression,which promotes the proliferation,migration and invasion of SACC cells.

OBJECTIVE: To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. METHODS: Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. RESULTS: After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. CONCLUSIONS TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.
Male ; Animals ; Swine ; Abdominal Injuries ; Apoptosis ; Cardiopulmonary Resuscitation ; Kidney Diseases

Spinal nerve injury causes mechanical allodynia and structural imbalance of neurotransmission, which were typically associated with calcium overload. Storeoperated calcium entry (SOCE) is considered crucial elements-mediating intracellular calcium homeostasis, ion channel activity, and synaptic plasticity. However, the underlying mechanism of SOCE in mediating neuronal transmitter release and synaptic transmission remains ambiguous in neuropathic pain. Neuropathic rats were operated by spinal nerve ligations. Neurotransmissions were assessed by whole-cell recording in substantia gelatinosa. Immunofluorescence staining of STIM1 with neuronal and glial biomarkers in the spinal dorsal horn. The endoplasmic reticulum stress level was estimated from qRT-PCR. Intrathecal injection of SOCE antagonist SKF96365 dose-dependently alleviated mechanical allodynia in ipsilateral hind paws of neuropathic rats with ED 50 of 18 μg. Immunofluorescence staining demonstrated that STIM1 was specifically and significantly expressed in neurons but not astrocytes and microglia in the spinal dorsal horn. Bath application of SKF96365 inhibited enhanced miniature excitatory postsynaptic currents in a dosage-dependent manner without affecting miniature inhibitory postsynaptic currents. Mal-adaption of SOCE was commonly related to endoplasmic reticulum (ER) stress in the central nervous system. SKF96365 markedly suppressed ER stress levels by alleviating mRNA expression of C/ EBP homologous protein and heat shock protein 70 in neuropathic rats. Our findings suggested that nerve injury might promote SOCE-mediated calcium levels, resulting in long-term imbalance of spinal synaptic transmission and behavioral sensitization, SKF96365 produces antinociception by alleviating glutamatergic transmission and ER stress. This work demonstrated the involvement of SOCE in neuropathic pain, implying that SOCE might be a potential target for pain management.

Objective:To investigate the clinical experience of different types of femoral perforator flaps in the reconstruction of oral and maxillofacial head and neck defects.Methods:From January 2018 to January 2021, 573 patients with oral and maxillofacial head and neck defects reconstructed by femoral perforator flap were collected in the Department of Maxillofacial Oncology, the Third Affiliated Hospital of Air Force Military Medical University (age range of 21-76 years, with a male to female ratio of 1.23∶1). According to the type of perforator flap, the patients were divided into ALT group, AMT group, TFL flap group and free muscle flap group. The incidence of postoperative complications, wound healing time and drainage volume in femoral area were compared among the 4 groups.Results:The ALT flap was used in 527 cases: 22 flaps had vascular crisis, 14 flaps had infection, 8 flaps had necrosis, 519 flaps survived; the mean healing time of the wound was (14.50±3.19) days, and the mean drainage volume was (49.9±21.3) ml. 28 cases were repaired with AMT flap: 2 flaps had vascular crisis and 1 had infection. All the flaps survived; the mean healing time of the wound was (14.18±2.75) days, and the mean drainage volume was (50.3±23.0) ml. 11 cases were repaired by TFL flap: 1 flap had vascular crisis and 1 had infection. All the flaps survived. The mean healing time of the wound was (14.09±2.66) days, and the mean drainage volume was (54.1±25.0) ml. 7 cases were repaired by free muscle flap survived without vascular crisis, infection and other postoperative complications; the mean healing time of the wound was 14.14±1.86, and the mean postoperative drainage volume was (49.9±21.1) ml. There was no significant difference in complication rate (flap necrosis, vascular crisis, infection, etc.) and repair effect among 573 patients with different flap types. The postoperative follow-up was conducted for 6-24 months, and the donor area was smooth and good in appearance, without obvious scar or functional influence. The repair effect of the affected area was satisfactory.Conclusions:Although there is a certain proportion of perforator vessel variation in the femoral perforator flap, the flap can be designed freely according to different types of variation. The thigh perforator flap has an essential application value in the repair of oral and maxillofacial head and neck defects.

Objective:To investigate the characteristics of executive function of preschool children with high functioning autism spectrum disorder (HFA) and with global developmental delay (GDD), and the differences among HFA, GDD and typically developmental (TD) children.Methods:From January 2020 to January 2021, 20 male HFA, 20 male GDD and 20 male TD children aged 4-6 years who visited the Psychological Behavior Clinic of the Child Health Department of Guiyang Maternal and Child Health Hospital and the Developmental Behavior Clinic of the Children Health Department of the Ninth People's Hospital in Chongqing were selected for comparative study.The executive function of HFA, GDD and TD children was assessed with the behavior rating scale of executive function-preschool version(BRIEF-P) and the executive function task program (EF-TOUCH). SPSS 26.0 software was used for statistical analysis, including variance test, independent sample t-test, χ2 test, Kruskal Wallis test and Mann-Whitney U test. Results:In the EF-TOUCH program task, the accuracy of the three groups of children's performance in the pig task (Pig), the silly sounds game (SSG), the working memory task (pick the picture, PTP) and the task of cognitive flexibility (something's the same, STS) were statistically different(Pig: HFA group: 0.87(0.76, 0.99), GDD group: 0.97(0.85, 0.99), TD group: 1.00(0.98, 1.00), χ2=15.646, P<0.001; SSG: HFA group: 0.76(0.53, 0.91), GDD group: 0.76(0.65, 0.99), TD group: 0.94(0.76, 1.00), χ2=6.448, P=0.040; PTP: HFA group: 0.66±0.18, GDD group: 0.66±0.19, TD group: 0.78±0.11; F=3.221, P=0.048; STS: HFA group: 0.67(0.63, 0.70), GDD group: 0.72(0.46, 0.78), TD group: 0.87(0.83, 0.90), χ2=26.898, P<0.001). The accuracies of Pig, SSG, PTP and STS in HFA group were significantly lower than those in TD group(all P<0.05), and the accuracies of Pig and STS in GDD group were significantly lower than those in TD group(both P<0.05). In inhibition control, there were statistically differences in response time of Pig and SSG among the three groups (Pig: HFA group: (1 694.36±222.83)ms, GDD group: (1 513.46±244.91)ms, TD group: (1 444.84±197.95)ms, F=5.810, P=0.005; SSG: HFA group: (2 202.42±195.58)ms, GDD group: (2 116.52±323.27)ms, TD group: (1 937.17±252.74)ms, Z=4.610, P=0.014). There were no significant differences in the reaction time of Arrows task ( P>0.05). There were significant differences in BRIEF-P inhibition control, organizational planning, inhibition self-regulation, cognitive flexibility and total scores among the three groups ( P<0.05), while there were no significant differences in the scores of other factors and dimensions ( P>0.05). Conclusion:The executive function of pre-school children with high functioning autism spectrum disorder and children with global developmental delay is impaired.The executive function of children with high functioning autism spectrum disorder and children with global developmental delay is significantly different from that of typically developmental children of the same age.Moreover, the executive function of children with HFA is more severely damaged from all components than that of children with GDD.

Osteoarthritis is a chronic inflammatory disease characterized by non inflammatory degeneration of articular cartilage and the formation of osteophytes at the edge of the joint, caused by complex causes. Its pathology is complex, and its pathogenesis is not yet clear, ultimately leading to joint stiffness and functional activity disorders. At present, the treatment for osteoarthritis is limited to alleviating symptoms and improving function, with varying degrees of side effects. Ferroptosis is a new type of programmed cell death discovered in recent years, which is related to the pathological and physiological processes of osteoarthritis and plays an important regulatory role in the occurrence and development of osteoarthritis. Its main characteristics include iron metabolism imbalance and accumulation of reactive oxygen species. Therefore, ferroptosis inhibitors targeting ferroptosis have shown great application prospects in the treatment of osteoarthritis. In this review, the author summarizes the relevant mechanisms of ferroptosis in the occurrence and development of osteoarthritis, outlines a large number of specific therapeutic drugs and their corresponding targets, with the aim of delaying and reversing the progression of osteoarthritis by regulating chondrocyte ferroptosis, which has certain clinical guiding significance.

Objective:To explore the effect of structured interdisciplinary bedside rounds (SIBR) model on diarrhea and nutritional status in patients with stroke receiving enteral nutrition through nasogastric tube.Methods:From February to September 2021, 117 stroke patients with enteral nutrition through nasogastric tube admitted to Taiyuan Central Hospital were selected as the study subject by convenient sampling. The patients were divided into the control group (57 cases) and the intervention group (60 cases) according to the ward at the time of admission. The control group received routine enteral nutrition intervention, and the intervention group carried out enteral nutrition intervention based on SIBR model. The incidence of diarrhea and incontinence dermatitis during hospitalization, the nutritional indicators before treatment and on the seventh and fourteenth days of nutritional treatment, the incidence of complications during hospitalization and the length of hospitalization were compared between the two groups.Results:The incidence, duration, severity of diarrhea and the incidence of incontinence dermatitis in the intervention group were lower than those in the control group, and the incidence of constipation, gastric retention and pulmonary infection were lower than those in the control group, and the length of hospitalization was less than that in the control group, and the differences were statistically significant ( P<0.05) . After 14 days of nutritional treatment, the albumin, total protein and hemoglobin in the intervention group were better than those in the control group, with statistical differences ( P<0.05) . Conclusions:SIBR model integrates the content of ward round, shortens the time of ward round, increases the work efficiency, effectively reduces the incidence of diarrhea and incontinence dermatitis in patients with stroke receiving enteral nutrition through nasogastric tube, improves the nutritional status of patients, reduces the incidence of complications, and shortens the length of hospitalization.