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Objective:To investigate changes in the peripheral interleukin-35 (IL-35) level in patients with alopecia areata, and to assess its modulatory effect on regulatory T (Treg) cell activities.Methods:Totally, 81 patients with alopecia areata (alopecia areata group) and 27 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Sera and peripheral blood mononuclear cells (PBMCs) were isolated. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum IL-35 level, real-time fluorescence-based quantitative PCR to determine the mRNA expression of IL-35 subunits EBI3 and IL-12p35, and flow cytometry to determine the proportion of CD4 + CD25 + CD127 dim/- Treg cells. Sorted Treg cells were stimulated by recombinant human IL-35, ELISA was performed to detect levels of perforin and granzyme B in the culture supernatant, and real-time fluorescence-based quantitative PCR to determine the mRNA expression of EBI3, IL-12p35, and immune checkpoint molecules, such as programmed death protein 1 (PD-1) , T cell immunoglobulin and mucin protein-3 (Tim-3) , cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in Treg cells. IL-35-stimulated or unstimulated Treg cells were co-cultured with autologous PBMCs, and cell counting kit-8 (CCK8) assay was conducted to assess cellular proliferative activity. Measurement data were compared between 2 groups by using t test, comparisons among multiple groups were carried out by using one-way analysis of variance, correlation analysis was carried out by using Pearson correlation analysis, and enumeration data were compared by using chi-square test. Results:Compared with the control group, the alopecia areata group showed significantly decreased IL-35 levels (90.10 ± 11.98 ng/L vs. 100.74 ± 28.71 ng/L, t= 2.71, P= 0.008) , mRNA expression of EBI3 and IL-12p35 in PBMCs (EBI3: 1.06 ± 0.15 vs. 1.25 ± 0.11, t= 6.09, P < 0.001; IL-12p35: 1.00 ± 0.15 vs. 1.38 ± 0.22, t= 10.16, P < 0.001) , and proportions of Treg cells (5.91% ± 1.17% vs. 6.85% ± 1.23%, t= 3.54, P= 0.001) . In the alopecia areata group, the proportion of Treg cells was positively correlated with the serum IL-35 level ( r= 0.25, P= 0.026) , and the mRNA expression of EBI3 and IL-12p35 in PBMCs ( r= 0.31, 0.24, P= 0.004, 0.032, respectively) . Compared with the control group, the unstimulated Treg cells from the alopecia areata group showed significantly decreased supernatant levels of perforin and granzyme B, mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules ( P < 0.05 or 0.001) , as well as weakened inhibitory effect on the proliferative activity of PBMCs ( P= 0.013) . There was no significant difference in the level of perforin or granzyme B between the recombinant human IL-35-stimulated and unstimulated Treg cells from the patients with alopecia areata (both P > 0.05) . However, the mRNA expression of EBI3, IL-12p35 and immune checkpoint molecules was significantly higher in the IL-35-stimulated Treg cells than in the unstimulated Treg cells in the alopecia areata group ( P < 0.05 or 0.001) , and the inhibitory effect on the proliferative activity of PBMCs was also significantly enhanced in the IL-35-stimulated Treg cells compared with the unstimulated Treg cells ( P= 0.037) . Conclusion:The peripheral IL-35 level was significantly decreased in the patients with alopecia areata, which was closely associated with reduced activities of Treg cells, and IL-35 may be involved in the occurrence of alopecia areata.
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Objective:To investigate changes of natural killer (NK) cell subsets and interleukin-18 (IL-18) level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity.Methods:A total of 67 patients with alopecia areata (alopecia areata group) and 25 healthy volunteers (control group) were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16 + NK cells. Comparisons between groups were performed using t test or paired t test. Results:Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56 +CD16 - NK cells (8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001) , but significantly increased percentage of CD56 +CD16 + NK cells (46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001) , and there was no significant difference in the percentage of CD56 -CD16 + NK cells between the alopecia areata group and control group (28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383) . The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group (112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015) . After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group (9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively) than in those from the control group (5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05) . After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16 + NK cells (151.10% ± 59.30%) compared with the control group (221.90% ± 93.56%, t = 4.31, P < 0.001) . After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system (14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001) , while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems (both P > 0.05) . Conclusion:IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.
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Objective To investigate the neuroprotective effect and its mechanism of p38 mitogenactivated protein kinase inhibitor after subarachnoid hemorrhage (SAH).Methods Twenty-seven SPF-grade adult male SD rats were selected to induce a SAH model using the prechiasmal pool blood injection.Three dead rats were excluded.Twenty-four rats were randomly divided into four groups:sham operation group,SAH group,dimethyl sulfoxide (DMSO) group,and DMSO +p38 inhibitor group (n =6 in each group).Western blot was used to detect the expression levels of p38,phosphorylation p38,Parkinson's disease protein 7 (DJ-1),autophagy associated gene 5 (Atg5),autophagy adaptor protein p62,microtubule-associated protein Ⅰ Light Chain 3 (LC3-Ⅰ),microtubule-associated protein Ⅱ light chain 3 Ⅱ (LC3-Ⅱ),and the Garcia neurological function score was used to judge the nerve injury.PC12 cell oxygenated hemoglobin was used to induce an in vitro SAH model.They were completely randomly divided into four groups:sham operative group,SAH group,DMSO group,and DMSO + p38 inhibitor group.Fluorescent probe JC-1 was used to observe the changes of mitochondrial membrane potential.Results (1) There were significant differences in the expression of p38,phosphorylation-p38 and DJ-1 in rat brain tissue among the 4 groups (F values were 94.959,150.293 and 698.476,respectively,all P ＜ 0.01).There were significant differences in mitochondrial membrane potential in PC12 cells among the 4 groups (F value was 24.989,P ＜ 0.01).There were significant differences in the expression levels of autophagy related protein LC3-Ⅱ/LC3-Ⅰ ratio,Atg5 and p62 in rat brain tissue among the 4 groups (F values were 235.319,110.490 and 36.311,respectively,all P ＜ 0.01).There was significant difference in nerve function score among the 4 groups (F value was 25.550,P ＜ 0.01).(2) Compared with the sham operative group,the expression levels of p38,phosphorylation-p38 and DJ-1 were upregulated significantly after SAH (from 0.43 ±0.06,0.41 ±0.02 and 0.07 ±0.01 to 0.61 ± 0.08,0.79 ± 0.07 and 0.17 ± 0.03,respectively,all P ＜ 0.01),mitochondria membrane potential depolarization (from 8.29 ±0.28 to 9.23 ±0.42,P ＜0.01);upregulation of Atg5 expression and increase of LC3-Ⅱ/LC3-Ⅰratio (from 0.23 ± 0.04 and 0.25 ± 0.04 to 0.47 ± 0.04 and 0.46 ± 0.04,respectively,all P ＜ 0.01),down regulation of p62 expression (from 1.09 ± 0.14 to 0.54 ± 0.10,P ＜ 0.01);neurological score was decreased (from 17.5 ± 0.6 to 11.3 ± 2.7,P ＜ 0.01);p38 inhibitor was significantly down regulated the expression of phosphorylation-p38 after SAH (from 0.79 ± 0.07 to 0.47 ± 0.04,P ＜ 0.01),the expression of DJ-1 was up-regulated (from 0.17 ± 0.03 to 1.02 ± 0.06,P ＜ 0.01),mitochondrial membrane potential recovery (from 9.23 ±0.42 to 8.47 ±0.36,P ＜0.01),cell autophagy related protein LC3-Ⅱ/LC3-Ⅰ ratio and Atg5 were upregulated(from 0.46 ±0.04 and 0.47 ±0.04 to 0.77 ± 0.06 and 0.95 ± 0.12,all P ＜ 0.01),p62 expression returned to the levels of SAH group (from 0.57 ± 0.09,to 0.54 ± 0.10,P =0.650),and the neurological score was significantly improved (from 11.3 ± 2.7 to 15.5 ± 1.0,P ＜0.01).Conclusions After SAH,the p38 inhibitor downregulates the activity of2 phosphorylation p38.It may inhibit abnormal autophagy and maintain mitochondrial function by up-regulating the expression of DJ-1 protein,and then play a neuroprotective function.
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Objective To investigate whether the monitoring of cerebral blood flow can be used to evaluate the successful preparation of rat models with focal cerebral ischemia.Methods With the line plug inserted into the left internal cerebral artery of 30 SPF Wistar Han rats at (16.0±0.5) mm,(18.0±0.5) mm and (20.0±0.5) mm respectively,three kinds of focal cerebral ischemia models (10 each) were prepared.All the rats were divided into incomplete occlusion group,complete occlusion group and deep occlusion group according to whether the blood clots were found in skull base and the infarction was appeared in the middle cerebral artery territory after the whole brain was stained with 2,3,5-triphenyl tetrazolium chloride.The cerebral blood flow in the middle cerebral artery territory of each animal was monitored by laser Doppler at the time of before and after blocking with the line plug inserted and reperfusion with the line plug pulled out and recorded for statistical analysis.The cerebral blood flow was expressed as the relative flow unit (perfusion unit,PU);The changes of the cerebral blood flow after occlusion and reperfusion were expressed as a percentage of that blood flow and those before occlusion.Results During the process of modeling,1 rats died,and the other in the incomplete occlusion group (n=9),in the complete occlusion group (n=15),and in the deep occlusion group (n=5).The depths of inserting of 8 rats in the incomplete occlusion group were about (16±0.5) mm,and the blood flow from the anterior cerebral artery to middle cerebral artery could not be prevented completely,the Longa score was evaluated 6 h after ischemia and ranged from 0 to 1,there were no blood clots in skull base and no infarction after TTC staining.The depths of inserting of 9 rats in the complete occlusion group were about (18±0.5) mm,and the anterior cerebral artery blood flow was completely blocked,the neurological dysfunction was significant after 6 h ischemia and the Longa score was ranged from 2 to 3,the blood clots in skull base were absent and the infarctions in middle cerebral artery territory were obvious after TTC staining.The depth of inserting of 5 rats in the deep occlusion group were about (20±0.5) mm,and the anterior cerebral artery blood flow was also completely blocked,the neurological dysfunction was serious after 6 h ischemia and the Longa score was ranged from 3 to 4,there existed blood clots in skull base and obvious infarctions in middle cerebral artery territory after TTC staining.The cerebral blood flows after the line plug inserted compared with those before occlusion in the incomplete occlusion group,complete occlusion group and deep occlusion group were all decreased (94±17 vs.256±36,43±9 vs.286±44,44±6 vs.294±46,respectively,all P0.05);The percentages of cerebral blood flow after occlusion and before were (36.93±0.06)%,(15.09±0.02)%,(15.52±0.04)% respectively,and the difference between groups was statistically significant (F=39.14,P<0.01).The cerebral blood flow after reperfusion were 213±31,147±17,96±14,respectively,and the difference was also statistically significant (F=50.05,P<0.01),and the cerebral blood flow in deep occlusion group was less than that in complete occlusion group(P<0.05);The percentages of cerebral blood flow after reperfusion and before occlusion were (83.10±0.02)%,(51.83±0.05)%,(33.49±0.09)% respectively,and the difference was also statistically significant (F=93.23,P<0.01).Conclusion The cerebral blood flow monitored by laser Doppler can be used as a real-time,convenient,micro invasive,objective and reliable standard to evaluate the successful preparation of rat MCAO models with line plug method.
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Objective To investigate the possible mechanism of hyperhomocysteinemia (HHcy) in renal dysfunction in patients with chronic stable coronary artery disease (SCAD).Methods We consequentially enrolled patients diagnosed as having SCAD and,according to homocysteine (Hcy) levels,divided them into the HHcy group (Hcy≥15 μmol/L,n =53) and control group (Hcy＜15 μmol/L,n =47).We further tested the relationship among plasma Hcy level,renal function,and serum levels of interleukin (IL)-6 and adiponectin (APN).Results IL-6 was significantly higher in the HHcy group than in the control group (P ＜ 0.05).However,APN level was obviously lower in the HHcy group than in the control group (P ＜ 0.05).Correlation analysis revealed that Hcy level positively correlated with creatinine (r =0.379,P ＜ 0.001),uric acid (r =0.238,P =0.019),cystatin C (r =0513,P ＜ 0.001),and IL-6 levels (r =0.561,P ＜ 0.001) but negatively correlated with estimated glomerular filtration rate (eGFR;r =-0.288,P =0.023) and APN level (r =-0.428,P ＜ 0.001).IL-6 level showed positive correlations with creatinine (r =0.406,P =0.002),uric acid (r =0.359,P =0.007),and cystatin C levels (r =0.387,P =0.007) but a negative correlation with eGFR (r =-0.370,P =0.005).Meanwhile,APN level showed negative correlations with creatinine (r =-0.694,P ＜ 0.001),uric acid (r =-0.420,P ＜0.001),and cystatin C levels (r =-0.553,P ＜ 0.001),but a positive correlation with eGFR (r =0.251,P =0.034).Conclusion Plasma Hcy level may have a predictive value for renal dysfunction in patients with SCAD.Moreover,HHcy probably promoted renal dysfunction by inducing the imbalance of anti-inflammation and pro-inflammation in patients with SCAD.
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Abstract A chiral separation and residue determination method for cis-epoxiconazole enantiomers in apple, grape and tea samples was developed and validated by ultra performance convergence chromatography combined with quadrupole time-of-flight mass spectrometry ( UPC2-QTOF/MS) . The Chrial CCA column was used to separate cis-epoxiconazole enantiomers and the chromatography conditions ( mobile phase modifier and proportion, column temperature, automated backpressure regulator, and auxiliary solvent ) were optimized. Samples were extracted by acetonitrile, and respectively purified by Cleanert TPT or Pesti-Carb solid phase extraction ( SPE ) columns, then analyzed by UPC2-QTOF/MS. The optimum conditions were as follows:mobile phase was CO2/isopropanol (95: 5, V/V), flow-rate was 2. 0 mL/min, automated backpressure regulator (ABPR) was 13. 79 MPa, column temperature was 30℃, with a post-column mauxiliary solvent of methanol/water (1:1, V/V) containing 2 mmol/L ammonium formate. The analyte was quantified by matrix external standard method. The results showed that linear range of this method was 0. 01-1. 00 mg/L, and the correlation coefficients were above 0 . 99 . The recoveries of cis-epoxiconazole enantiomers at three spiked levels (0. 005, 0. 025 and 0. 25 mg/kg) in fruit matrix were 67. 9%-92. 8% with relative standard deviations (RSDs, n=6) less than 10%, and the limit of quantification (LOQ) of enantiomers was 0. 005 mg/kg. The recoveries of cis-epoxiconazole enantiomers at three spiked levels (0. 01, 0. 05 and 0. 5 mg/kg) in black tea were 74 . 1% -84 . 0% with RSDs ( n=6 ) less than 8%, and the LOQ for these two enantiomers was 0. 01 mg/kg. This method is rapid, convenient and reliable, and could meet the requirement of residue analysis.
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Objective To explore the surgical treatment of posttraumatic epilepsy in functional cerebral area Methods After preoperative evaluation,nineteen patients with intractable posttraumatic epilepsy of functional cerebral area underwent multiple subpial transaction under the electrocorticogram monitoring during the operation, combining with anterior corpus callosotomy, anterior temporal lobectomy and selective amygdalo-hippocampectomy. Results The follow-up results showed seizures of these patients improved considerably. According to Engel' s grading,among 19 cases,10 cases were grade Ⅰ ,7 cases were grade Ⅱ ,2 cases were grade Ⅲ ,and no cases were grade Ⅳ. Conclusions The satisfactory clinical outcome of the surgical treatment of intractable posttraumatic epilepsy in functional cerebral area could be obtained with the help of careful preoperative evaluation and multiple subpial transaction under the electrocorticogram monitoring.
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Objective To discuss the methods of treatment of sellar region tumors by microsurgery via modified transpterional approach.Methods The clinical data of 326 patients with sellar region tumors were retrospectively analyzed. All of the patients(of them 158 patients with pituitary adenomas, 89 patients with craniopharyngiomas and 79 patients with meningiomas)were treated by microsurgery via modified transpterional in our hospital in recent 4 years. The most important part of preoperative arrangement was giving glucocorticoid and anti-epilepsy drugs. Modified transpterional craniotomy was performed, and the tumors were removed by employing microsurgical techniques via the 5 anatomy interspaces in sellar region after the subarachnoid cisterns were dissected carefully under surgical microscope. Consciousness, urine volume and serum electrolyte of the patients were observed strictly after surgery, and the complications such as diabetes insipidus, serum electrolyte disorder and epilepsy were remedied in time.Results The rate of intact frontal branch of facial nerve was 92.18% and that of intact supraorbital nerve was 95.72%. Atrophy of temporal muscle occurred in none of the patients. Total and/or subtotal resection of tumors was achieved in 95.26% patients and the rate of intact pituitary stalk was 62.44%. The major postoperative complications included diabetes insipidus and serum electrolyte disorder. Although the incidence of complications was high to 61.42%, complete restore was achieved in 97.12% of the patients with the complications within 1 month.Conclusion The modified transpterional approach for sellar region tumors may give a lower incidence of damage to frontal branch of facial nerve, super-orbital nerve and temporal muscle, and it can achieve higher total resection rate and a less postoperative complications by employing modified transpterional approach and the microsurgical techniques, combined with the active treatments of postoperative complications, therefore it is deserved to be spread to use in clinical works.
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Objective To investigate the methods of total resection of craniopharyngioma and treatment of postoperative complications. Methods The abscission of CPG was performed in 24 patients with craniopharyngioma (CPG) which situated at sellar region after giving glucocorticoid and anti-epilepsy drug. The tumors were removed via the 4 anatomy spaces in sellar region by employing microsurgical technique. Consciousness, urine, and serum electrolyte of patients were observed strictly after surgery, and complications such as diabetes insipidus, serum electrolyte disorders and epilepsy were remedied in time. Results Total removal of tumors were achieved in 22 cases, and minor portion of tumors was left in 2 cases. All of the patients were sober after surgery. Diabetes insipidus, serum electrolyte disorder and body temperature disorder took place in 16 cases,12 cases, and 6 cases respectively. After treatment, serum electrolyte disorder and body temperature disorder were rectified in all cases, but diabetes insipidus was rectified in 13 of 16 cases. Conclusion Choosing the best operation approach according to the scope which tumor invades and being familiar with the 4 sellar anatomy spaces so as to be used correctly in operation are the keys to remove the CPG completely. Giving glucocorticoid before operation and active treatment of complications such as diabetes insipidus, serum electrolyte disorder can decrease mortality observably.
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Objective To investigate the method of total resection of craniopharyngioma and prevention and treatment of post-operative complications. Methods Pterion craniotomy was performed in 24 patients with craniopharyngioma which was situated in sellar region after giving glucocorticoid and anti-epilepsy drug. The tumors were removed via the 4 anatomy spaces in sellar region by employing microsurgical technique. Consciousness, urine volume, and serum electrolyte of patients were observed strictly after surgery, and complications such as diabetes insipidus, serum electrolyte disorders and epilepsy were treated in time. Results Total removal of tumors were achieved in 22 cases, and minor portion of tumors was left in 2 cases. There was not obvious deficit of nervous system function in all of the patients after surgery. Diabetes insipidus, serum electrolyte disorder and body temperature disorder occurred in 16 cases, 12 case, and 6 cases respectively. After treatment, serum electrolyte disorder and body temperature disorder were rectified in all cases, and diabetes insipidus was rectified in 13 of 16 cases. Conclusion Choosing the best operation approach according to the scope of tumor invasion and being familiar with the 4 sellar anatomy spaces were the keys to remove craniopharyngioma completely. Giving glucocorticoid before operation and active treatment of complications such as diabetes insipidus and serum electrolyte disorder could effectively decrease mortality.
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Objective To investigate the clinical and pathological features of Wegener granulomatosis to improve the management of it. Methods Twenty-three patients with Wegener granulomatosis were analyzed retrospectively. Results The average age at onset was 37.2 years (range from 20~57 years). The average interval from clinical symptoms presentation to diagnosis was 5.8 months. The initial symptom of 23 cases was the upper respiratory tract involvement (48%). The patients had multiple system and organ involvement. 87% patients with WG had lung involvement and 78% patients had glucocorticoids. Seventeen out of 23 were cANCA (74%) positive. The pathological manifestations included necrotizing granulomatosis and vasculitis. Conclusion Clinical manifestation of WG is complicate, including multiple system and organ involvement. The upper and lower respiratory tract and kidney are the most commonly involved organs. Necrotizing granulomatosis and vasculitis are the pathological features of WG.
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Objective To evaluate the method of measurement of total scatter calibrate factor (Sc,p). Methods To measure the Sc,p at different depths on central axis of 6?MV, 15?MV photon beams through different ways. Results It was found that the measured data of Sc,p changed with the different depths to a range of 1%～7%. Using the direct method, the Sc,p measured depth should be the same as the depth in dose normalization point of the prescription dose. If the Sc,p ( fsz, d) was measured at the other depths, it could be obtained indirectly by the calculation formula. Conclusions The Sc,p in the prescription dose can be obtained either by the direct measure method or the indirect calculation formula. But emphasis should be laid on the proper measure depth.