Yao R Q, Ren C, Wang L X, Dong N, Wu Y, Yao Y M
Trauma Research Center, Fourth Medical Center and Medical Innovation Research Department of PLA General Hospital, Beijing 100048, China.
Zhonghua Shao Shang Za Zhi. 2020 Aug 20;36(8):658-664. doi: 10.3760/cma.j.cn501120-20200430-00246.
To explore the influence of Xuebijing injection (hereinafter referred to as Xuebijing) and its component paeoniflorin on immune function of regulatory T cells (Tregs) of spleen and survival rate of septic rats. (1) CD4(+) CD25(+) Tregs and CD4(+) T cells were isolated and purified from spleens of three 9 to 12 weeks old Sprague-Dawley male rats (the same age, breed, and gender below) by immunomagnetic beads. According to the random number table (the same grouping method below), CD4(+) CD25(+) Tregs were divided into blank control group, simple CD3/CD28 group, simple endotoxin/lipopolysaccharide (LPS) group, LPS+ Xuebijing group, and LPS+ paeoniflorin group, with 6 wells in each group. The cells in simple CD3/CD28 group, simple LPS group, LPS+ Xuebijing group, and LPS+ paeoniflorin group were cultured in RPMI 1640 medium containing fetal bovine serum in volume fraction of 10%, 1.25 μg CD3, and 2.5 μg CD28 for 24 hours. Then 1 μg/mL LPS in the volume of 1 μL was added to the cells in simple LPS group, LPS+ Xuebijing group, and LPS+ paeoniflorin group. Moreover, 5 mg/mL Xuebijing in the volume of 1 μL and 80 μmol/L paeoniflorin in the volume of 1 μL were added to the cells in LPS+ Xuebijing group and LPS+ paeoniflorin group, respectively, which were cultured for another 72 hours. Cells in blank control group were routinely cultured in RPMI 1640 medium containing fetal bovine serum in volume fraction of 10% for 96 hours. The expressions of cytotoxic T lymphocyte antigen 4 (CTLA-4) and forkhead wing-link transcription factor 3 (Foxp3) and apoptosis of CD4(+) CD25(+) Tregs were measured by flow cytometry. The interleukin-10 (IL-10) level from culture supernatant of CD4(+) CD25(+) Tregs was determined by enzyme-linked immunosorbent assay (ELISA). CD4(+) T cells were divided into blank control' group, simple CD3/CD28' group, simple LPS' group, LPS+ Xuebijing' group, and LPS+ paeoniflorin' group, with 6 wells in each group. After being cocultured with the corresponding CD4(+) CD25(+) Tregs treated as before for 72 hours, the proliferative activity of CD4(+) T cells was measured by flow cytometry, and IL-4 level from culture supernatant of CD4(+) T cells was determined by ELISA. (2) One hundred and twenty rats were divided into sham surgery group, simple sepsis group, sepsis+ Xuebijing group, and sepsis+ paeoniflorin group, with 30 rats in each group. The septic rat model was reproduced by cecal ligation and puncture surgery in simple sepsis group, sepsis+ Xuebijing group, and sepsis+ paeoniflorin group. In sham surgery group, the rats were only performed with laparotomy to simulate surgery. In sepsis+ Xuebijing group, the rats were given post-surgical injection of 4 mL/kg Xuebijing through tail vein, twice a day. In sepsis+ paeoniflorin group, the rats received 978 μg paeoniflorin via tail vein, twice a day. The survival rates of rats in the four groups on post surgery day 1, 2, 3, 4, 5, 6, and 7 were observed and recorded. The surviving cure of Kaplan-Meier was drawn. Data were statistically analyzed with one-way analysis of variance, least significant difference test. The surviving curve was analyzed by Log-rank (Mantel-Cox) test. (1) Compared with those in blank control group, the expressions of CTLA-4 and Foxp3 of CD4(+) CD25(+) Tregs (=27.19, 17.00, <0.01) and IL-10 level from culture supernatant (=40.76, <0.01) were significantly increased in rats in simple LPS group. Compared with those in simple LPS group, the expressions of CTLA-4 and Foxp3 of CD4(+) CD25(+) Tregs ((LPS+ Xuebijing group)=31.03, 11.27, (LPS+ paeoniflorin group)=5.79, 5.64, <0.01) and IL-10 level from culture supernatant (=15.49, 4.20, <0.01) was significantly decreased in LPS+ Xuebijing group and LPS+ paeoniflorin group. Compared with that in blank control group, the apoptosis rate of CD4(+) CD25(+) Tregs in simple LPS group was significantly declined (=6.02, <0.01). Compared with the rate in simple LPS group, the apoptosis rates of CD4(+) CD25(+) Tregs in LPS+ Xuebijing group and LPS+ paeoniflorin group were significantly increased (=20.32, 8.60, <0.01). (2) Compared with those in simple CD3/CD28' group, the proliferative rate of CD4(+) T cells was significantly decreased in simple LPS' group (=22.47, <0.01), while IL-4 level from culture supernatant was significantly elevated (=3.51, <0.01). Compared with those in simple LPS' group, the proliferative rates of CD4(+) T cells in LPS+ Xuebijing' group and LPS+ paeoniflorin' group were significantly increased (=16.31, 11.48, <0.01), while IL-4 level from culture supernatant showed no obvious change. (3) The post-operative 7-day survival rates of rats in sham surgery group, simple sepsis group, sepsis+ Xuebijing group, sepsis+ paeoniflorin group were 100% (30/30), 30% (9/30), 57% (17/30), and 47% (14/30), respectively. Compared with that in simple sepsis group, the survival rate within post-operative 7-day of rats in sepsis+ Xuebijing group was significantly higher ((2)=4.34, <0.05), while the survival rate within post-operative 7-day of rats in sepsis+ paeoniflorin group showed no obvious change. Both Xuebijing and its component paeoniflorin are capable of reversing sepsis-induced inhibitory immune function and apoptotic resistant of Tregs in rats, and further improving the proliferative activity of T cells. In addition, the effect of paeoniflorin on improvement of survival rate of rats with sepsis is weaker than Xuebijing.
探讨血必净注射液（以下简称血必净）及其成分芍药苷对脓毒症大鼠脾脏调节性T细胞（Tregs）免疫功能及生存率的影响。（1）采用免疫磁珠法从3只9至12周龄的Sprague-Dawley雄性大鼠（下同，年龄、品种、性别相同）脾脏中分离纯化CD4(+)CD25(+)Tregs和CD4(+)T细胞。根据随机数字表（下同分组方法），将CD4(+)CD25(+)Tregs分为空白对照组、单纯CD3/CD28组、单纯内毒素/脂多糖（LPS）组、LPS+血必净组、LPS+芍药苷组，每组6孔。单纯CD3/CD28组、单纯LPS组、LPS+血必净组、LPS+芍药苷组细胞在含体积分数为10%胎牛血清、1.25μg CD3和2.5μg CD28的RPMI 1640培养基中培养24小时。然后向单纯LPS组、LPS+血必净组、LPS+芍药苷组细胞中加入1μL体积的1μg/mL LPS。此外，分别向LPS+血必净组和LPS+芍药苷组细胞中加入1μL体积的5mg/mL血必净和1μL体积的80μmol/L芍药苷，再培养72小时。空白对照组细胞在含体积分数为10%胎牛血清的RPMI 1640培养基中常规培养96小时。采用流式细胞术检测CD4(+)CD25(+)Tregs中细胞毒性T淋巴细胞抗原4（CTLA-4）、叉头翼状螺旋转录因子3（Foxp3）的表达及凋亡情况。采用酶联免疫吸附测定（ELISA）法检测CD4(+)CD25(+)Tregs培养上清液中白细胞介素-10（IL-10）水平。将CD4(+)T细胞分为空白对照组、单纯CD3/CD28'组、单纯LPS'组、LPS+血必净'组、LPS+芍药苷'组，每组6孔。与相应的经上述处理的CD4(+)CD25(+)Tregs共培养72小时后，采用流式细胞术检测CD4(+)T细胞的增殖活性，采用ELISA法检测CD4(+)T细胞培养上清液中IL-4水平。（2）将120只大鼠分为假手术组、单纯脓毒症组、脓毒症+血必净组、脓毒症+芍药苷组，每组30只。单纯脓毒症组、脓毒症+血必净组、脓毒症+芍药苷组采用盲肠结扎穿孔术复制脓毒症大鼠模型。假手术组大鼠仅行剖腹术模拟手术。脓毒症+血必净组大鼠术后经尾静脉注射4mL/kg血必净，每日2次。脓毒症+芍药苷组大鼠经尾静脉注射978μg芍药苷，每日2次。观察并记录4组大鼠术后第1、2、3、4、5、6、7天的生存率。绘制Kaplan-Meier生存曲线。数据采用单因素方差分析、最小显著差法进行统计学分析。生存曲线采用Log-rank（Mantel-Cox）检验进行分析。（1）与空白对照组相比，单纯LPS组大鼠CD4(+)CD25(+)Tregs的CTLA-4、Foxp3表达（=27.19，17.00，<0.01）及培养上清液IL-10水平（=40.76，<0.01）显著升高。与单纯LPS组相比，LPS+血必净组、LPS+芍药苷组CD4(+)CD25(+)Tregs的CTLA-4、Foxp3表达（（LPS+血必净组）=31.03，11.27，（LPS+芍药苷组）=5.79，5.64，<0.01）及培养上清液IL-10水平（=15.49，4.20，<0.01）显著降低。与空白对照组相比，单纯LPS组CD4(+)CD25(+)Tregs的凋亡率显著下降（=6.02，<0.01）。与单纯LPS组相比，LPS+血必净组、LPS+芍药苷组CD4(+)CD25(+)Tregs的凋亡率显著升高（=20.32，8.60，<0.01）。（2）与单纯CD3/CD28'组相比，单纯LPS'组CD4(+)T细胞的增殖率显著降低（=22.47，<0.01），而培养上清液IL-4水平显著升高（=3.51，<0.01）。与单纯LPS'组相比，LPS+血必净'组、LPS+芍药苷'组CD4(+)T细胞的增殖率显著升高（=16.31，11.48，<0.01），而培养上清液IL-4水平无明显变化。（3）假手术组、单纯脓毒症组、脓毒症+血必净组、脓毒症+芍药苷组大鼠术后7天生存率分别为100%（30/30）、30%（9/30）、57%（17/30）、47%（14/30）。与单纯脓毒症组相比，脓毒症+血必净组大鼠术后7天生存率显著升高（（2）=4.34，<0.05），而脓毒症+芍药苷组大鼠术后7天生存率无明显变化。血必净及其成分芍药苷均能逆转脓毒症诱导的大鼠Tregs免疫功能抑制及凋亡抵抗，并进一步提高T细胞增殖活性。此外，芍药苷改善脓毒症大鼠生存率的作用弱于血必净。
