Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey . Methods To detect the expression of P21 gene in COS-7 cells ( derived from the kidney of African green monkey , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern blot assay .Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA.Their silencing rate was (91.82 ±3.21)%, (82.47 ±2.48)%, (81.31 ±2.69 )% and ( 87.35 ±4.59 )%, and the protein expression was ( 11.97 ±0.70 )%, ( 20.22 ±0.65 )%, ( 23.21 ± 0.63)%and (14.42 ±0.86)%, respectively.Conclusions Four effective silencing target sequences are screened at cel-lular level , which can be used in gene silencing research of rhesus monkeys .

OBJECTIVETo verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.

METHODSImmunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.

RESULTSCASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.

CONCLUSIONCASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

Cell Proliferation ; genetics ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; metabolism ; Fibroblasts ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction

Objective:To explore the protective effect of vagus nerve stimulation (VNS) on the prognosis of rats suffering from cardiac arrest/cardiopulmonary resuscitation (CA/CPR) under different treatment timings.Methods:The method of percutaneous epicardial electrical stimulation was used to establish CA model of rat. Fifty-three male SD rats were randomly (random number) divided into the sham group ( n=5), CPR group ( n=12), PRE group ( n=12), POST5 group ( n=12) and POST30 group ( n=12). The sham group did not experience CA/CPR. VNS treatment was started at 30 min before CA (PRE group, n=12), 5 min after recovery of spontaneous circulation (ROSC) (POST5 group, n=12), and 30 min after ROSC (POST30 group, n=12) in different VNS-treated group, respectively. The electrical stimulation was applied to the vagus nerve for 30 min with a unified parameter. The neurological deficit scores at 24, 48, and 72 h after ROSC were recorded, and the survival rate in each group was observed. TUNEL staining was used to detect the apoptosis of cortical area and the expression of α7 nicotinic acetylcholine receptor (α7nAChR) in brain tissue was measured by immunofluorescence at 72 h after ROSC. Variables were compared with one-way analysis of variance, and survival for Kaplan-Meier curves were tested with the log-rank test. A P value less than 0.05 was considered statistically significant. Results:Compared with the CPR group (survival rate 33.33%), both pre-treatment (survival rate 75%) and post-treatment of VNS (POST5 group survival rate 75% and POST30 group survival rate 83.33%) significantly improved the 72 h survival rate after CPR ( P<0.05), mitigated neurological deficits after ROSC, reduced the positive rate of apoptosis neurons, and up-regulated the expression of α7nAChR in cerebral cortex. There was no significant difference among the VNS-treated groups (all P>0.05). Conclusions:Both pre-treatment and post-treatment of VNS can play a protective role in rats after CA/CPR, which may be related to the activation of α7nAChR-mediated anti-inflammatory and anti-apoptosis effects.

Objective To investigate the role of TLR4/NF-κB signaling pathway under the action of TAK-242 in the cardiomyocyte apoptosis after coronary micro-embolism (CME) in rats.Methods Fortyfive rats were randomized (random number) into three groups:sham operation,CME and CME plus TAK242 groups (n =15 per group).CME was induced by injecting polyethylene microspheres (42 μm) into the left ventricle except the sham group.CME plus TAK-242 group was treated with TAK-242 (2 mg/kg) via the tail vein of mice 30 min before CME modeling.Cardiac function was evaluated 6 h after operation.Tissue biopsy was stained with HBFP to measure the size of infarction area.TUNEL assay was used to detect cardiomyocyte apoptosis.Western blot and qPCR were used to evaluate the protein levels and mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3,respectively.Statistical analysis was performed using one-way analysis of variance followed by LSD-t test.Results Compared with the sham group,left ventricular ejection fraction (LVEF) in the CME group was significantly decreased [(68.91 ± 4.12) ％ vs.(84.80 ± 2.51) ％,P ＜ 0.05],and the infarction area (P ＜ 0.05),the apoptosis index [(3.36 ± 0.63) ％ vs.(0.19 ± 0.08) ％,P ＜0.05],the mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3 in CME group were increased significantly (all P ＜ 0.05).Compared with CME group,LVEF in the CME plus TAK-242 group was significantly improved [(75.58 ± 5.01) ％ vs.(68.91 ± 4.12) ％,P＜0.05],and the infarction area [(8.58 ± 2.12) ％ vs.(14.65 ± 4.23) ％,P＜0.05],the apoptosis index [(1.43 ± 0.51) ％ vs.(3.36 ± 0.63) ％,P ＜ 0.05],the mRNA expressions of TLR4,NF-κB p65 and cleaved caspase-3 in CME + TAK-242 group were decreased significantly (all P ＜ 0.05).Conclusions TAK-242 effectively improved CME-induced cardiac dysfunction by regulating TLR4/NF-κB signaling pathway and then reducing the cardiomyocyte apoptosis.

Objective In order to establish a rhesus monkey model of p53 gene silencing, firstly we screened and determined the effective silencing targets of p53 gene at the cellular level in rhesus monkey.Methods The expression of p53 gene was detected in COS-7 cells ( derived from the kidney of the African Green Monkey, Cercopithecus aethiops).Three small hairpin RNA ( shRNA) sequences targeting rhesus monkey p53 gene were designed, analysed by bioinformatics, and inserted into lentivirus-based gene silencing constructs FUGW-TDT.The plasmids of p53-RNAi and control vector were transfected into the COS-7 cells, respectively.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western blot assay.Results p53 gene expression was detected in COS-7 cells.Bioinformatics analysis showed that three gene-silencing sequences were screened which lied in the open reading frame ( ORF) region and targeted 238 -258bp, 681 -701bp, 169 -189bp of the rhesus monkey p53 mRNA.At 48 hrs after transfection of the three silencing constructs, p53 mRNA was suppressed by(87.17 ±4.03)%, ( 72.62 ±4.11)% and(76.22 ±0.98 )%, and p53 protein was suppressed by ( 84.44 ±2.18 )%, ( 71.04 ±1.18)% and ( 74.17 ±0.95 )%, respectively. Conclusions We obtained three effective target sequences showing high efficiency in p53silencing, which can be used in further studies on gene silencing in rhesus monkey.

Objective:To investigate the effect of botulinum neurotoxin type A (BoNT/A) on Nav1.3 sodium channels and the function of sodium current in neuropathic pain.Methods:Eighteen adult rats had a spared nerve injury (SNI) induced and then were randomly divided into a saline group and a BoNT/A group, each of 9. Another 9 rats formed a fake operation group, Five days after the SNI, BoNT/A (7U/kg or 15U/kg) or saline was subcutaneously administered into the plantar surface of a hindpaw. On the 3rd, 7th and 14th day after the injection, the SNI rats′ paw withdrawal threshold was measured. On the 7th and 14th day the expression of Nav1.3 protein in dorsal root ganglion (DRG) neurons was examined using western blotting, while any change in the functional tetrodotoxin-sensitive (TTX-S) current was recorded using the patch clamp technique.Results:Administration of BoNT/A at either 7U/kg or 15U/kg significantly increased the SNI-induced mechanical allodynia. The expression of Nav1.3 protein in DRG neurons increased significantly after the SNI, but had decreased significantly by the 7th and 14th day after the administration of BoNT/A. BoNT/A significantly decreased the current density in TTX-S sodium channels in DRG neurons following SNI.Conclusion:Administration of BoTN/A affects the expression of Nav1.3 protein and functional TTX-S current, relieving neuropathic pain.

Objective:To investigate the clinicopathological features, treatment programs and prognosis of patients with primary diffuse large B-cell lymphoma (DLBCL) in cavernous sinus.Methods:The clinical data of a patient with primary DLBCL in cavernous sinus who were admitted to Wuhan No.1 Hospital in December 2020 were retrospectively analyzed, and the relevant literature was reviewed.Results:The patient was a 63-year-old female who underwent resection of the cavernous sinus lesion, and the pathological diagnosis was DLBCL. The patient received 6 courses of R-CHOP regimen chemotherapy, lumbar puncture + intrathecal injection of chemotherapy drugs, and twice additional rituximab immunochemotherapy, and no tumor cells were found in the results of liquid-based thin layer cytology for cerebrospinal fluid exfoliated cells; twice magnetic resonance imaging (MRI) re-examination after the operation showed no recurrence and adjacent metastasis of the tumor. The patient's symptoms were significantly improved without residual neurological sequelae.Conclusions:Primary DLBCL in cavernous sinus is rare in clinical practice, early diagnosis is crucial for the prognosis of patients, and different protein expression may indicate the prognosis. Biopsy, complete resection of the tumor under the premise of preserving important anatomical structures and functions, and standardized chemotherapy combined with intrathecal injection local chemotherapy can effectively prolong the survival time of patients and improve the quality of life.

Electroacupuncture treatment of primary insomnia is widely used and with confirmed efficacy. The factors influencing the therapeutic effect of electroacupuncture include waveform selection, treatment frequency, stimulation intensity, stimulation time and acupoint selection. The mechanism of electroacupuncture in treating this disease mainly includes regulating the levels of excitatory neurotransmitters, γ-aminobutyric acid, melatonin and interleukin cytokines.

Objective To noninvasively predict isocitrate dehydrogenase(IDH)status of glioma via combining imaging and clini-cal features before surgery,so as to provide basis for individualized clinical treatment decision.Methods A total of 47 patients with glioma confirmed by pathological and molecular genetic tests were included,including 20 with IDH mutant type and 27 with IDH wild type.After diffusion tensor imaging(DTI)scanning,fractional anisotropy(FA)and mean diffusivity(MD)values of tumor paren-chyma were calculated.Combining DTI parameters with MRI morphological features of tumor,blood neutrophil/lymphocyte ratio(NLR)and patient's age,binary logistic regression model was established to effectively predict IDH status of glioma patients before surgery.Results There were significant differences in FAmean/FANAWM,MDmin,NLR,tumor location and age between IDH mutant type and IDH wild type groups(P<0.05).The binary logistic regression model concluding,FAmean/FANAWM,MDmin,cystic degeneration,NLR and age,predicted IDH status of glioma with area under the curve(AUC)of 0.961 and 95%confidence interval(CI)of 0.914-1.00.Conclusion The regression model established via combining DTI,MRI morphological features and blood NLR has great performance in classifying IDH status of glioma,and can help predict IDH status noninvasively before surgery,so as to assist clinical individualized treatment.

Objective To explore the clinical efficacy of ginseng schisandra decoction in children with lobar pneumonia(lung spleen deficiency syndrome)induced by mycoplasma pneumoniae infection and improvement of inflammatory indicators.Method 84 children with lobar pneumonia(lung spleen deficiency syndrome)caused by mycoplasma infection admitted to the First Clinical Hospital of Jilin Academy of Traditional Chinese Medicine from January 2020 to July 2022 were selected and randomly divided into two groups with 42 cases each.The conventional group was treated according to the traditional treatment plan,while the combined group was treated with ginseng and schisandra decoction in addition to the traditional treatment plan.After 7 days,compare the differences in traditional Chinese medicine syndrome scores,clinical efficacy,inflammatory indicators,and total incidence of adverse reactions between the two groups of children.Result The results showed that the traditional Chinese medicine syndrome score and inflammatory indicators in the combined group were lower than those in the conventional group(P<0.05),and the clinical efficacy level distribution was better than that in the conventional group(P<0.05).The total effective rate was higher than that in the conventional group(P<0.05),and there was no difference in the total incidence of adverse reactions between the two groups(P>0.05).Conclusion In the treatment of children with lobar pneumonia(lung spleen deficiency syndrome)caused by Mycoplasma pneumoniae infection,the modified Ginseng Wuweizi decoction has a positive therapeutic effect and good safety,and is worth promoting.