Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

Objective:To evaluate the effect of α-ketoglutarate (AKG) on postoperative delirium (POD) in aged mice.Methods:Eighty male C57BL/6N mice, aged 18 months, weighing 30-35 g, were divided into 4 groups ( n=20 each) using a random number table method: control+ solvent group (group C), control+ AKG group (group C+ AKG), surgery+ solvent group (group S) and surgery+ AKG group (group S+ AKG). Dimethyl 2-oxoglutarate 0.6 mg/kg was injected intraperitoneally for 3 consecutive days before surgery in C+ AKG and S+ AKG groups, while the equal volume of normal saline was given in C and S groups.Exploratory laparotomy was performed under anesthesia with isoflurane to establish POD model.The behaviors of mice in each group were tested at 24 h before surgery and 6, 9 and 24 h after surgery using buried food test (the latency to eat food), open field test (total distance, latency to the center, time and freezing time spent in the center) and Y maze test (duration in the novel arm and the number of entries into the novel arm), respectively.Then the animals were sacrificed at 6 h after operation, hippocampal tissues were removed for determination of the expression of microglia-specific marker ionized calcium binding adaptor molecule-1 (Iba-1), the number of Iba-1 positive cells (using immunofluorescence staining), and the expression of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in hippocamapus (by Western blot). Results:Compared with group C, the latency to eat food at eath time point was significantly prolonged, latency to the center at 6 and 9 h after surgery was prolonged, time spent in the center at 6 and 9 h after surgery was shortened, freezing time at 6, 9 and 24 h after surgery was shortened, the number of entries into the novel arm at 6 and 9 h after surgery was decreased, duration in the novel arm at 6 h after surgery was shortened, the expression of Iba-1 was up-regulated, the number of Iba-1 positive cells was increased, and the expression of IL-1β and TNF-α in hippocampus was up-regulated in group S ( P<0.05), and no significant change was found in the behaviors indexes in group C+ AKG ( P>0.05). Compared with group S, the latency to eat food at each time point was significantly shortened, latency to the center at 9 h after surgery was shortened, time spent in the center at 6 and 9 h after surgery was prolonged, freezing time at 9 and 24 h after surgery was prolonged, the number of entries in the novel arm at 9 h after surgery was increased, the expression of Iba-1was down-regulated, the number of Iba-1 positive cells was decreased, and the expression of IL-1β and TNF-α in hippocampus was down-regulated in group S+ AKG ( P<0.05). Conclusion:AKG can alleviate POD, and the mechanism may be related to inhibiting the activation of microglia and and thus reducing inflammatory responses in aged mice.

This study was a single-center large-sample case-control study.Data of 1 106 elderly patients who underwent unilateral total hip arthroplasty from June 2013 to May 2019 were collected, including items such as patient′s baseline characteristics, comorbidities, perioperative medication, intraoperative blood pressure, and postoperative outcomes.Patients were divided into postoperative nausea and vomiting(PONV)group and non-PONV group according to whether nausea and vomiting occurred within 24 h after operation.Logistic regression analysis was used to determine the risk factors for PONV.The incidence of PONV was 11.03%.Female, intraoperative use of dezocine, and intraoperative hypotension(duration>3 min or cumulative time>6 min)are independent risk factors for PONV, while femoral neck fractures and intraoperative use of dexamethasone are protective factors.

Objective To investigate the expression of insulin-like growth factors-1(IGF-1)in ser-um and phosphorylated IGF-1 receptor in spinal cord in mouse model of bone cancer pain. Methods Sixty male C3H/HeJ mice weighed 18-22 g were randomly divided into Sham group(n=30)and Tumor group(n=30). The mice in Tumor group were inoculated with NCTC fibrosarcoma cells in the right femur bone marrow cavity. Paw withdrawl mechanical threshold(PWMT)and the number of spontaneous flinches(NSF)were measured on 1d before inoculation and on 4 d,7 d,10 d,14 d,21 d after inoculation(n=8). At each time point,the mice of each group were taken blood by removal eyeball and the samples of blood were obtained to detect the expression of IGF-1 by enzyme-linked immunosorbent assay(n=4). The mice after taken blood on 14 d after inoculation were perfused and the samples of spinal cord lumber(L3~5)segment were obtained to detect the expression of phosphorylated IGF-1 receptor by immunofluorescence assay(n=6). Results Com-pared with Sham group,PWMT was significantly decreased(P<0.05)and NSF was significantly increased(P<0.05)on 7~21 d after inoculation. Compared with baseline value and Sham group(baseline value(27.33± 0.52)pg/ml,Sham group(29.11±1.86)pg/ml,(24.51±3.61)pg/ml,(23.33±4.59)pg/ml,(25.29±2.99) pg/ml),the expression of IGF-1 in serum was significantly increased on 7~21 d after inoculation in Tumor group((39.76±3.92)pg/ml,(36.93±2.18)pg/ml,(38.85±2.40)pg/ml,(39.70±2.62)pg/ml). The mean fluorescence intensity of phosphorylated IGF-1 receptor was significantly higher on 14d after inoculation in Tumor group(2.40±0.11)compared with Sham group(0.05±0.01). Conclusion Expression of IGF-1 in serum and phosphorylated IGF-1 receptor in spinal cord were significantly increased in mice with bone cancer pain,and this change may be involved in the development and maintenance of bone cancer pain.

To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP).
 Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490.
 Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05).
 Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.
Animals ; Astrocytes ; pathology ; Bone Neoplasms ; complications ; Hyperalgesia ; drug therapy ; etiology ; Injections, Spinal ; Male ; Mice ; Mice, Inbred C3H ; Rats, Sprague-Dawley ; Spinal Cord ; cytology ; pathology ; Tyrphostins ; administration & dosage

Objective To evaluate the effect of verapamil on the expression of K+-Cl-cotransporter 2 (KCC2) in spinal dorsal horns during remifentanil-induced hyperalgesia in a rat model of incisional pain.Methods Thirty-two pathogen-free healthy adult male Sprague-Dawley rats,aged 6-7 weeks,weighing 250-300 g,were divided into 4 groups (n=8 each) using a random number table:control group (group C),incisional pain group (group Ⅰ),incisional pain plus remifentanil plus verapamil group (group I+R+ V) and incisional pain plus remifentanil group (group I+R).Normal saline was subcutaneously infused in group C.A 1 cm long incision was made in the plantar surface of the right hindpaw in anesthetized rats in group Ⅰ.Verapamil 5 mg/kg was intraperitoneally injected at 10 min before establishment of the incisional pain model in group I+R+V.In I+R and I+R+V groups,the model of incisional pain was established,and remifentanil was subcutaneously infused for 30 min at a rate of 80 μg · kg-1 · h-1 simultaneously.The mechanical paw withdrawal threshold (MWT) to yon Frey filament stimulation was measured at 1 day before establishment of the model (T0) and 2,6,24 and 48 h after establishment of the model (T1-4).The rats were sacrificed after measurement of MWT at T4,and the lumbar enlargement segments of the spinal cord were harvested for determination of the expression of KCC2 by immunofluorescence.Results Compared with group C,the MWT was significantly decreased at T1-4,and the expression of KCC2 was down-regulated in the other groups (P＜0.05).Compared with group Ⅰ,the MWT was significantly decreased at T1-4,and the expression of KCC2 was down-regulated in group I+R (P＜0.05).Compared with group I+R,the MWT was significantly increased at T1-4,and the expression of KCC2 was up-regulated in group I+R+V (P＜0.05).Conclusion The mechanism by which verapamil reduces remifentanil-induced hyperalgesia is related to up-regulation of the expression of KCC2 in spinal dorsal horns in a rat mnodel of incisional pain.

Objective To evaluate the role of spinal astrocytes in posttraumatic stress disorder (PTSD)-induced hyperalgesia in rats. Methods Forty pathogen-free healthy male Sprague-Dawley rats, aged 6-8 weeks,weighing 200-250 g, were divided into 4 groups(n=10 each)using a random number table:control group(group C), group PTSD, normal saline group(group NS)and fluorocitrate group (group FC).The rats were exposed to single prolonged stress for establishment of the PTSD model in PTSD, NS and FC groups. At 30 min before establishment of the model and 1-7 days after establishment of the model,normal saline 10 μl was intraperitoneally injected in group NS, and 1 nmol∕10 μl fluorocitrate 10 μl, an inhibitor of astrocyte activation, was intraperitoneally injected in group FC. The mechanical paw withdrawal threshold(MWT)was measured at 24 h before establishment of the model and on 1, 2, 3, 5 and 7 days after establishment of the model. Four rats were sacrificed after measurement of pain threshold on 1 and 7 days after establishment of the model, and the lumbar segment(L3-5)of the spinal cord was re-moved for determination of the expression of glial fibrillary acidic protein(GFAP, an astrocyte marker)u-sing Western blot. Results Compared with group C, the MWT was significantly decreased at each time point after establishment of the model,and the expression of spinal GFAP was up-regulated on 1 and 7 days after establishment of the model in PTSD,NS and FC groups(P<005). Compared with group PTSD, no significant change was found in the MWT at each time point in group NS(P>005),and the MWT was sig-nificantly increased at each time point after establishment of the model,and the expression of spinal GFAP was down-regulated on 1 and 7 days after establishment of the model in group FC(P<005).Conclusion Activation of spinal astrocytes is involved in PTSD-induced hyperalgesia in rats.

Objective To evaluate the effects of different levels of neuromuscular blockade(NMB)on transcranial electric motor-evoked potentials(TCeMEPs)during idiopathic scoliosis.Methods Thirty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients of both sexes,aged 11-23 yr,weighing 31-62 kg,scheduled for elective idiopathic scoliosis under general anesthesia,were enrolled in the study.NMB was monitored with train of four(TOF)-Watch SX.The levels of partial NMB were classified into 5 states according to TOF ratio(TOFR)and TOF counts:1 or 2 TOF counts(TOF1),3 TOF counts and TOFR≤15%(TOF2),TOFR 16%-25%(TOF3),TOFR 26%-50%(TOF4),TOFR 51%-75%(TOF5) and TOFR>75%(no NMB).Each state was maintained for 10 min.Failure and false-positive findings in TCeMEP monitoring,development of unexpected body movement and satisfaction with NMB were recorded.Results Compared with no NMB,the failure and false-positive rates of TCeMEP monitoring were significantly increased,the incidence of unexpected body movement was decreased,and the rate of satisfactory NMB was increased at TOF1,TOF2 and TOF3(P<0.05),no significant change was found in failure or false-positive rates of TCeMEP monitoring at TOF4 and TOF5(P>0.05),and the incidence of unexpected body movement was decreased and the rate of satisfactory NMB was increased at TOF4,the rate of satisfactory NMB was increased at TOF5(P<0.05),and no significant change was found in the incidence of unexpected body movement at TOF5(P>0.05).Compared with those at TOF4,no significant change was found in the failure or false-positive rates of TCeMEP monitoring(P>0.05),the incidence of unexpected body movement was significantly increased,and the rate of satisfactory NMB was decreased at TOF5(P<0.05).Conclusion Maintaining TOFR at 26%-50% the partial NMB during surgery does not affect TCeMEP monitoring during idiopathic scoliosis and meets the intra-operative NMB requirements simultaneously,and it is the optimum NMB for this type of surgery.

Objective To investigate the relationship between spinal neuronal microRNA 212 (miR-212) and phosphorylation of cAMP response element-binding protein (CREB) in a mouse model of bone cancer pain (BCP).Methods Thirty-two male SPF C3H/HeJ mice, aged 4-6 weeks, weighing 20-25 g, were randomly divided into 4 groups (n=8 each) using a random number table: sham operation group (group S), BCP group, BCP + intrathecal negative control locked nucleic acid (LNA) group (group BC) , and BCP + intrathecal miR-212 antisense LNA group (group BL).After the mice were anesthetized with intraperitoneal pentobarbital sodium, 20 μl of α minimal essential medium containing NCTC 2472 cells 2×105 was injected directly into the medullary cavity of the distal femur.In BC and BL groups, negative control LNA and miR-212 antisense LNA 12 pmol/5 μl were intrathecally injected, respectively, once a day for 7 consecutive days, starting from day 14 after inoculation.In S and BCP groups, the equal volume of DNAse/RNAse-free water was given instead.The number of spontaneous flinches (NSF) and mechanical paw withdrawal threshold (MWT) were measured on 1 day before inoculation and 4, 7, 10, 14 and 21 days after inoculation.The mice of each group were sacrificed after measurement of pain threshold on 21 days after inoculation, and the lumbar enlargement segments of the spinal cord were harvested to detect the expression of phosphorylated CREB (p-CREB) and CREB using Western blot.Results Compared with group S, the MWT was significantly decreased, and the NSF was increased on 7-21 days after inoculation, and the expression of p-CREB was up-regulated in BCP, BC and BL groups.Compared with group BCP, the MWT was significantly increased, and the NSF was decreased on 21 days after inoculation, and the expression of p-CREB was down-regulated in group BL, and no significant change was found in the parameters mentioned above in BC group.There was no significant difference in the expression of CREB between the four groups.Conclusion Spinal neuronal miR-212 is involved in the maintenance of BCP probably by promoting phosphorylation of CREB in mice.

OBJECTIVE:To explore the content variation rule and influencing factors of 5-hydroxymethylfurfural(5-HMF)in the extraction process of the herbs Schisandra chinensis and improve the quality of monitoring the extract of Schisandra chinensis. METHODS:High performance liquid chromatography was adopted to determine the contents of 5-HMF in the extracts during the extraction process (decoction,vacuum concentration,alcohol precipitation,vacuum drying,alkali adjustment),and the content variation rule was found out. For vacuum drying during which the content of 5-HMF reduced obviously,the effects of the tempera-ture and time of drying on the content of 5-HMF were studied,and the effects of vacuum drying and freeze drying on the 5-HMF contents were compared. RESULTS:The content variation trend in the extraction process of Schisandra chinensis was as follows as a large amount of 5-HMF was produced in the herbs decoction process;the content of 5-HMF increased during concentration,re-duced during the alcohol precipitation and the vacuum drying of the extracts by three times concentrate,and maintained substantial-ly unchanged during alkali adjustment. In the process of vacuum drying,the content of 5-HMF decreased with temperature rise and time extension. Freeze drying had no effect as good as that of vacuum drying in the reduction in the content of 5-HMF. CON-CLUSIONS:It is suggested that the vacuum drying of the extracts by three times concentrate at higher temperature,which may pro-duce the extract of Schisandra chinensis with lower content of 5-HMF and thus improve the quality of Schisandra chinensis extract.