Objective:To investigate the effects of stimulator of interferon gene (STING) on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 (ACSL4) in mouse dendritic cells (DCs) under sepsis, providing a basis for improving the dysregulation of immune response in sepsis caused by factors such as wound infection.Methods:This study was an experimental research. The mouse DC line DC2.4 in the logarithmic growth phase (with passages 3-10) were divided into lipopolysaccharide (LPS) stimulation 0 h (unstimulated) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 18 h group, and LPS stimulation 24 h group according to the random number table (the same grouping method below), which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of phosphorylated STING (p-STING), STING, and ACSL4 in cells were determined by Western blotting. DC2.4 successfully transfected with lentivirus containing STING gene small interfering RNA (hereinafter referred to as siSTING) were divided into siSTING+phosphate buffer solution (PBS) group and siSTING+LPS group. DC2.4 successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After being stimulated with PBS or LPS and cultured for 24 hours, the protein expressions of p-STING, STING, and ACSL4 in cells were determined as above. Cell lipid peroxidation degrees were observed using the lipid peroxidation assay kit, and cell apoptosis rates were detected using flow cytometry. The sample numbers in the above cell experiments were all 3. Eighty male C57BL/6J mice aged 6 to 8 weeks were divided into sham surgery+normal saline (NS) group, cecal ligation and puncture (CLP)+NS group, sham surgery+C-176 group, and CLP+C-176 group, with 20 mice in each group. Mice in the two C-176 groups were intraperitoneally injected with C-176, while mice in the two NS groups were intraperitoneally injected with an equivalent volume of NS. One hour later, sham surgery was performed on the mice in the two sham surgery groups, and CLP surgery was performed on the mice in the two CLP groups to establish a sepsis model. At 24 h post-surgery, 10 mice from each group were sacrificed to extract spleen DCs, and protein expression, lipid peroxidation, and apoptosis rates were detected as above ( n=3). Hematoxylin-eosin staining was performed to observe pathological damage in the heart, liver, lung, and kidney tissue. The remaining 10 mice in each group were observed for survival within 7 days after surgery. Results:The protein expressions of p-STING, STING, and ACSL4, as well as the p-STING/STING ratio in DC2.4 in LPS stimulation 24 h group were significantly higher than those in LPS stimulation 0 h group ( P<0.05). After 24 h of culture, the protein expressions of p-STING, STING, and ACSL4 in DC2.4 in siSTING+LPS group and empty vector+PBS group were significantly lower than those in empty vector+LPS group ( P<0.05); the lipid peroxidation degrees of DC2.4 in siSTING+LPS group and empty vector+PBS group were weaker than those in empty vector+LPS group. The apoptosis rates of DC2.4 in empty vector+PBS group, empty vector+LPS group, siSTING+PBS group, and siSTING+LPS group were (15.7±3.0)%, (37.8±2.9)%, (13.1±2.1)%, and (20.6±1.8)%, respectively. The apoptosis rates of DC2.4 in empty vector+PBS group and siSTING+LPS group were significantly lower than that in empty vector+LPS group ( P<0.05). At 24 h post-surgery, the protein expressions of p-STING and ACSL4, and the p-STING/STING ratio in spleen DCs of mice in CLP+NS group were significantly higher than those in sham surgery+NS group and CLP+C-176 group ( P<0.05); the protein expression of STING in spleen DCs of mice in CLP+NS group was significantly higher than that in sham surgery+NS group ( P<0.05); the lipid peroxidation degrees of spleen DCs of mice in CLP+C-176 group and sham surgery+NS group were weaker than that in CLP+NS group. The apoptosis rates of spleen DCs of mice in sham surgery+NS group and CLP+C-176 group were significantly lower than that in CLP+NS group ( P<0.05), and the apoptosis rate of spleen DCs of mice in CLP+C-176 group was significantly higher than that in sham surgery+C-176 group ( P<0.05). Pathological tissue damage in the heart, liver, lung, and kidney of mice in CLP+NS group was significantly worse than that in sham surgery+NS group, while such damage in the above organs of mice in CLP+C-176 group was significantly alleviated compared with that in CLP+NS group. The survival ratio of mice in CLP+NS group within 7 days after surgery was significantly lower than that in sham surgery+NS group ( χ2=8.30, P<0.05). Conclusions:Under sepsis, STING activation in mouse DCs is significant, which enhances ACSL4-mediated ferroptosis. Inhibiting STING activation can significantly reduce ACSL4-mediated ferroptosis level in mouse DCs under sepsis, thereby improving the survival rate of septic mice.

Objective:To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor.Methods:The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining.Results:Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. Conclusion:SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.

Objective:To investigate the influence of family with sequence similarity 134, member B (FAM134B)-mediated reticulophagy on lipopolysaccharide (LPS)-induced apoptosis of mouse dendritic cells (DCs), so as to provide a basis for improving the immune suppression of sepsis caused by wound infection and other factors.Methods:The experimental research methods were used. The DC line DC2.4 of the 3 rd to 10 th passage in the logarithmic growth stage was collected for experiments. DCs were divided into LPS stimulation 0 h (no stimulation) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group, which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of FAM134B, microtubule-associated protein 1 light chain 3B (LC3B), and transporter protein SEC61B were determined by Western blotting, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated ( n=3). DCs were divided into phosphate buffer solution (PBS) group and LPS group for corresponding treatment. After 24 hours of culture, the expression of FAM134B and its co-localization with lysosomal probes and LC3B were detected using immunofluorescence method, while the number of autolysosomes in cells were observed through transmission electron microscope. DCs were divided into the FAM134B-knockdown group that were transfected with lentivirus containing small interfering RNA (siRNA) sequence of FAM134B gene and the empty vector group with empty lentivirus transfected. At post transfection hour 72, the fluorescence expression of cells was observed under the inverted fluorescence phase contrast microscope, meanwhile, the normally cultured DCs were set as blank control group, and the same observation was performed at the corresponding time point. DCs were divided into PBS alone group and LPS alone group, DCs successfully transfected with lentivirus containing siRNA sequence of FAM134B gene were divided into FAM134B-knockdown+PBS group and FAM134B-knockdown+LPS group, and DCs successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. These cells were stimulated correspondingly and cultured for 24 hours. The protein expression of FAM134B was detected using Western blotting ( n=3); the apoptotic rate of cells was determined by flow cytometry ( n=3); the situation of apoptosis was observed by Hoechst staining, and the apoptotic rate was calculated ( n=5); the protein expressions of cleaved cysteine aspartic acid specific protease-3 (caspase-3), B cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were detected using Western blotting, and the ratio of Bax/Bcl-2 was calculated ( n=5). Data were statistically analyzed with one-way analysis of variance (ANOVA), least significant difference test, and ANOVA for factorial design. Results:Compared with those in LPS stimulation 0 h group, the protein expressions of FAM134B of cells in LPS stimulation 12 h group and LPS stimulation 24 h group were significantly increased ( P<0.05), the protein expressions of SEC61B of cells in LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group were significantly decreased ( P<0.05), and the ratios of LC3B-Ⅱ/LC3B-Ⅰ of cells in LPS stimulation 24 h group and LPS stimulation 72 h group were obviously increased ( P<0.05). As the most significant changes of three proteins were seen in the cells of LPS stimulation 24 h group, 24 h was used as the duration of subsequent LPS stimulation. After 24 hours of culture, the expression of FAM134B and its co-localization with LC3B and lysosomal probes in the cells of LPS group were all significantly enhanced, with a significant increase in the number of autolysosomes in comparison with those in PBS group. Both the empty vector group and the FAM134B-knockdown group showed high intensity fluorescence in the cells at post transfection hour 72, but the blank control group showed no fluorescence in the cells at the corresponding time point. After 24 hours of culture, the protein expression of FAM134B of cells in FAM134B-knockdown+PBS group was significantly lower than the expressions in PBS alone group and empty vector+PBS group (with P values all <0.05), the protein expression of FAM134B of cells in FAM134B-knockdown+LPS group was significantly lower than the expressions in LPS alone group and empty vector+LPS group (with P values all <0.05), the protein expression of FAM134B of cells in LPS alone group was significantly higher than that in PBS alone group ( P<0.05), while the protein expression of FAM134B of cells in empty vector+LPS group was significantly higher than that in empty vector+PBS group ( P<0.05). After 24 hours of culture, flow cytometry assay revealed that the apoptotic rate of cells in PBS alone group, LPS alone group, empty vector+PBS group, empty vector+LPS group, FAM134B-knockdown+PBS group, and FAM134B-knockdown+LPS group were (13.3±0.8)%, (32.6±4.3)%, (17.0±1.5)%, (51.7±3.3)%, (52.4±3.1)%, and (62.3±2.6)%, respectively. After 24 hours of culture, compared with those in LPS alone group and empty vector+LPS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in FAM134B-knockdown+LPS group ( P<0.05); compared with those in the corresponding PBS treatment group, namely, PBS alone group, empty vector+PBS group, and FAM134B-knockdown+PBS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in LPS alone group, empty vector+LPS group, and FAM134B-knockdown+LPS group ( P<0.05). Conclusions:The activation of reticulophagy mediated by FAM134B in mouse DCs is enhanced and peaked in 24 hours under LPS stimulation, and the activated reticulophagy has a significant inhibitory effect on cell apoptosis.

Objective:To investigate the role and significance of NUFIP-1-mediated ribophagy in apoptosis of dendritic cells (DCs) stimulated by lipopolysaccharide (LPS).Methods:Cultured mouse dendritic cell line DC2.4 were divided into the blank control group and LPS stimulation groups for 6, 12, 24, 48 and 72 h ( n=5). LPS subgroups were consistently cultured with 1 μg/mL LPS for the corresponding incubation time. Western blot was adopted to detect the expression levels of NUFIP-1 and autophagy-related proteins p62 and LC3B across groups. Laser scanning confocal microscopy (LSCM) was applied to detect the expression and cellular localization of NUFIP-1, with its co-localization with Lyso-tracker and LC3B, respectively. The silencing blank vector NS and silencing virus vector NUFIP-1 siRNA were transferred into DC2.4 ( n=3) and stimulated with 1 μg/mL LPS for 24 h. The apoptosis of DC2.4 was measured by flow cytometry analysis. The expression levels of apoptosis-related proteins were determined using Western blot, including cleaved caspase-3 and Bcl-2. One-way analysis of variance (ANOVA) was applied for comparison among multiple groups, and LSD-t method was used for subsequent pairwise comparison. A P<0.05 was considered statistically significant. Results:The results of Western blot showed that expression level of NUFIP-1 in DC2.4 revealed a trend of first increasing and subsequent decreasing upon LPS stimulation for different times (6, 12, 24, 48 and 72 h), and the expression level of NUFIP-1 in the LPS 24 h group was significantly higher than that in the blank control group [blank control group: (0.6786 ± 0.0820); LPS 24 h group: (1.4830 ± 0.1170); P<0.01]. Meanwhile, p62 expression in the LPS 24 h group was significantly lower than that in the blank control group [blank control group: (0.9087 ± 0.1235); LPS 24 h group: (0.3113 ± 0.5571); P<0.01]. Moreover, the conversion from LC3B-I to LC3B-II in the LPS 24 h group was significantly higher than that in the blank control group [blank control group: (0.5542 ± 0.1248); LPS 24 h group: (2.5310 ± 0.3119); P<0.01]. LSCM indicated that NUFIP-1 was predominantly located in the nucleus and perinuclear area in DC2.4. The fluorescence intensity of NUFIP-1 increased in a time-dependent manner from 6 h to 24 h after LPS stimulation, whereas a significant reduction could be observed at 48 h and 72 h after LPS stimulation. Meanwhile, the co-localization of NUFIP-1 with Lyso-tracker and LC3B was substantially reinforced in comparison with the blank control group. Transfection of NUFIP-1 siRNA through lentivirus transfection technology significantly down-regulated the expression level of NUFIP-1 in DC2.4, with statistical differences compared with the blank control group and empty vector group [blank control group: (0.6627 ± 0.1707); empty vector group: (0.6966 ± 0.1107); siRNA group: (0.1428 ± 0.0296); P<0.05]. Flow cytometry analysis revealed that the apoptotic rate of LPS-stimulated DC2.4 was significantly higher in the NUFIP-1 siRNA transfection group than that in the blank control group and empty vector group [blank control LPS 24 h group: (47.91% ± 1.006%); empty vector LPS 24 h group: (70.26% ± 1.011%); siRNA LPS 24 h group: (80.23% ± 2.094); P<0.01]. Western blot analysis of apoptosis-related protein further confirmed that the expression level of cleaved caspase-3 was significantly elevated in the NUFIP-1 siRNA transfection group compared to those of the blank control group and empty vector group under LPS challenge [blank control LPS 24 h group: (0.4748 ± 0.0876); empty vector LPS 24 h group: (0.2849 ± 0.0418); siRNA LPS 24 h group: (0.9733 ± 0.0525); P<0.01]. Likewise, expression of Bcl-2, an anti-apoptotic protein was significantly down-regulated in the siRNA LPS 24 h group [blank control LPS 24 h group: (0.7810 ± 0.0490); empty vector LPS 24 h group: (0.8292 ± 0.0729); siRNA LPS 24 h group: (0.3957 ± 0.0838); P<0.05]. Conclusions:NUFIP-1-mediated ribophagy is significantly activated in DC2.4 upon LPS stimulation, exerting an underlying protective effect on apoptosis.

Objective:To explore the effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats.Methods:① Assessment of coagulation: 144 male Sprague-Dawley (SD) rats were divided into four groups by random number table: sham group, cecal ligation and puncture (CLP) induced sepsis model group (CLP group), CLP+Xuebijing group, and CLP+hydroxysafflor yellow A group, with 36 rats in each group. CLP was used for reproducing septic models. The cecum of the rats in the sham group was exposed by laparotomy and then returned to the abdominal cavity without CLP, while the other steps were the same as those in the CLP group. Rats in the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group were injected with Xuebijing (4 mL/kg, twice a day) or hydroxysafflor yellow A solution (0.378 g/L, 298 μg each time, twice a day) through caudal vein after operation. Levels of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), and D-dimer in peripheral blood were measured by automatic coagulation analyzer at 6, 12, 24 hours after operation. The enzyme linked immunosorbent assay (ELISA) was applied to determine levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and soluble thrombomodulin (sTM) in peripheral blood. ② Analysis of survival rates: 120 rats were divided into four groups by random number table (the same groups with those in the section of assessment of coagulation), with 30 rats in each group. The Kaplan-Meier survival curve was plotted, and the cumulative survival rates were observed and recorded for 7 days after CLP surgery.Results:① Results of coagulation assessment: compared with the sham group, septic rats in the CLP group showed significant dysfunction in coagulation early, as evidenced by prolonged PT at 6 hours after CLP (s: 8.9±0.2 vs. 8.4±0.4, P < 0.01), and significantly increased levels of Fib, D-dimer, TFPI and sTM [Fib (g/L): 2.8±0.3 vs. 2.3±0.1, D-dimer (ng/L): 1.8±0.2 vs. 1.5±0.1, TFPI (ng/L): 131.1±10.9 vs. 102.8±10.5, sTM (μg/L): 27.2±1.2 vs. 19.8±2.9, all P < 0.01]. The coagulation dysfunction became more and more serious at 12 hours after operation, and further deteriorated with time. The use of both Xuebijing and hydroxysafflor yellow A revealed significant improvement in coagulation of septic rats at 6 hours, as shown by shortened PT (s: 8.3±0.2, 8.3±0.1 vs. 8.9±0.2, both P < 0.01), and decreased Fib, D-dimer, TFPI and sTM as compared with those in the CLP group [Fib (g/L): 2.3±0.1, 2.3±0.2 vs. 2.8±0.3; D-dimer (ng/L): 1.5±0.1, 1.5±0.2 vs. 1.8±0.2; TFPI (ng/L): 109.5±10.2, 91.5±5.0 vs. 131.1±10.9; sTM (μg/L): 22.3±1.5, 21.1±1.8 vs. 27.2±1.2; all P < 0.01]. However, there was no significant difference in coagulation function between the two intervention groups. ② Results of survival rates analysis: the rats in the sham group all survived 7 days after operation. The 7-day cumulative survival rate of the CLP group was only 36.67% (11/30). Compared with the CLP group, the cumulative survival rates were significantly increased in rats of the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group [66.67% (20/30), 66.67% (20/30) vs. 36.67% (11/30), both P < 0.05], but no significant difference was found between the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group. Conclusion:Both Xuebijing and its component hydroxysafflor yellow A appear to be capable of alleviating coagulation disorders and improving survival rates of septic rats effectively, and the effects show no significant difference between them.

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 ( tLPS+ Xuebijing group=31.03, 11.27, tLPS+ 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.

Although the clinical definitions of sepsis and recommended treatments are regularly updated, a systematic review has not been done for preclinical models. To address this deficit, a Wiggers-Bernard Conference on preclinical sepsis modeling reviewed the 260 most highly cited papers between 2003 and 2012 using sepsis models to create a series of recommendations. This PartⅡreport provides recommendations for the types of infections and documentation of organ injury in preclinical sepsis models. Concerning the types of infections, the review showed that the cecal ligation and puncture model was used for 44% of the studies while 40% injected endotoxin. Recommendation #8 (numbered sequentially from PartⅠ): endotoxin injection should not be considered as a model of sepsis; live bacteria or fungal strains derived from clinical isolates are more appropriate. Recommendation #9: microorganisms should replicate those typically found in human sepsis. Sepsis-3 states that sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection, but the review of the papers showed limited attempts to document organ dysfunction. Recommendation #10: organ dysfunction definitions should be used in preclinical models. Recommendation #11: not all activities in an organ/system need to be abnormal to verify organ dysfunction. Recommendation #12: organ dysfunction should be measured in an objective manner using reproducible scoring systems. Recommendation #13: not all experiments must measure all parameters of organ dysfunction, but investigators should attempt to fully capture as much information as possible. These recommendations are proposed as "best practices" for animal models of sepsis.

Preclinical animal studies are mandatory before new treatments can be tested in clinical trials. However, their use in developing new therapies for sepsis has been controversial because of limitations of the models and inconsistencies with the clinical conditions. In consideration of the revised definition for clinical sepsis and septic shock (Sepsis-3), a Wiggers-Bernard Conference was held in Vienna in May 2017 to propose standardized guidelines on preclinical sepsis modeling. The participants conducted a literature review of 260 most highly cited scientific articles on sepsis models published between 2003 and 2012. The review showed, for example, that mice were used in 79% and euthanasia criteria were defined in 9% of the studies. PartⅠof this report details the recommendations for study design and humane modeling endpoints that should be addressed in sepsis models. The first recommendation is that survival follow-up should reflect the clinical time course of the infectious agent used in the sepsis model. Furthermore, it is recommended that therapeutic interventions should be initiated after the septic insult replicating clinical care. To define an unbiased and reproducible association between a new treatment and outcome, a randomization and blinding of treatments as well as inclusion of all methodological details in scientific publications is essential. In all preclinical sepsis studies, the high standards of animal welfare must be implemented. Therefore, development and validation of specific criteria for monitoring pain and distress, and euthanasia of septic animals, as well as the use of analgesics are recommended. A set of four considerations is also proposed to enhance translation potential of sepsis models. Relevant biological variables and comorbidities should be included in the study design and sepsis modeling should be extended to mammalian species other than rodents. In addition, the need for source control (in case of a defined infection focus) should be considered. These recommendations and considerations are proposed as "best practices" for animal models of sepsis that should be implemented.

Preclinical animal studies are mandatory before new treatments can be tested in clinical trials. However, their use in developing new therapies for sepsis has been controversial because of limitations of the models and inconsistencies with the clinical conditions. In consideration of the revised definition for clinical sepsis and septic shock (Sepsis-3), a Wiggers-Bernard Conference was held in Vienna in May 2017 to propose standardized guidelines on preclinical sepsis modeling. The participants conducted a literature review of 260 most highly cited scientific articles on sepsis models published between 2003 and 2012. The review showed, for example, that mice were used in 79% and euthanasia criteria were defined in 9% of the studies. PartIof this report details the recommendations for study design and humane modeling endpoints that should be addressed in sepsis models. The first recommendation is that survival follow-up should reflect the clinical time course of the infectious agent used in the sepsis model. Furthermore, it is recommended that therapeutic interventions should be initiated after the septic insult replicating clinical care. To define an unbiased and reproducible association between a new treatment and outcome, a randomization and blinding of treatments as well as inclusion of all methodological details in scientific publications is essential. In all preclinical sepsis studies, the high standards of animal welfare must be implemented. Therefore, development and validation of specific criteria for monitoring pain and distress, and euthanasia of septic animals, as well as the use of analgesics are recommended. A set of four considerations is also proposed to enhance translation potential of sepsis models. Relevant biological variables and comorbidities should be included in the study design and sepsis modeling should be extended to mammalian species other than rodents. In addition, the need for source control (in case of a defined infection focus) should be considered. These recommendations and considerations are proposed as "best practices" for animal models of sepsis that should be implemented.
Animals ; Mice ; Models, Animal ; Research Design ; Sepsis ; Shock, Septic

Although the clinical definitions of sepsis and recommended treatments are regularly updated, a systematic review has not been done for preclinical models. To address this deficit, a Wiggers-Bernard Conference on preclinical sepsis modeling reviewed the 260 most highly cited papers between 2003 and 2012 using sepsis models to create a series of recommendations. This Part II report provides recommendations for the types of infections and documentation of organ injury in preclinical sepsis models. Concerning the types of infections, the review showed that the cecal ligation and puncture model was used for 44% of the studies while 40% injected endotoxin. Recommendation #8 (numbered sequentially from Part I): endotoxin injection should not be considered as a model of sepsis; live bacteria or fungal strains derived from clinical isolates are more appropriate. Recommendation #9: microorganisms should replicate those typically found in human sepsis. Sepsis-3 states that sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection, but the review of the papers showed limited attempts to document organ dysfunction. Recommendation #10: organ dysfunction definitions should be used in preclinical models. Recommendation #11: not all activities in an organ/system need to be abnormal to verify organ dysfunction. Recommendation #12: organ dysfunction should be measured in an objective manner using reproducible scoring systems. Recommendation #13: not all experiments must measure all parameters of organ dysfunction, but investigators should attempt to fully capture as much information as possible. These recommendations are proposed as "best practices" for animal models of sepsis.
Animals ; Cecum ; Disease Models, Animal ; Endotoxins ; Humans ; Models, Animal ; Multiple Organ Failure ; Sepsis