Influence of Xuebijing injection and its component paeoniflorin on immune function and survival rate of septic rats

Objective 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. Methods (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 t test. The surviving curve was analyzed by Log-rank (Mantel-Cox) test. Results (1) Compared with those in blank control group, the expressions of CTLA-4 and Foxp3 of CD4⁺ CD25⁺ Tregs (t=27.19, 17.00, P<0.01) and IL-10 level from culture supernatant (t=40.76, P<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 (t_{LPS+ Xuebijing group}=31.03, 11.27, t_{LPS+ paeoniflorin group}=5.79, 5.64, P<0.01) and IL-10 level from culture supernatant (t=15.49, 4.20, P<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 (t=6.02, P<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 (t=20.32, 8.60, P<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 (t=22.47, P<0.01), while IL-4 level from culture supernatant was significantly elevated (t=3.51, P<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 (t=16.31, 11.48, P<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 (χ²=4.34, P<0.05), while the survival rate within post-operative 7-day of rats in sepsis+ paeoniflorin group showed no obvious change. Conclusions 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.