Objective:To design and evaluate a cell membrane vaccine strategy based on dendritic cell membrane microbubbles(DCM@MBs),and to explore its potential application in tumor immunotherapy,especially the immune-specific killing of tumor cells through the activation of T cells.Methods:At first,tumor cell membrane proteins were extracted and dendritic cells(DCs)were acti-vated to confirm that tumor antigens could effectively stimulate the maturation of immature DCs.Mature DC membranes were then mixed with lipids to prepare DCM@MBs,which were characterized for morphology,size,and protein composition by confocal laser scanning microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Finally,in vitro co-culture experiments were con-ducted to assess the effect of DCM@MBs on the activation of T cells and their ability for specific killing of tumor cells.Results:In the in vitro DC activation experiment,after stimulation with tumor cell membrane proteins,the 25 μg/mL group had a significant increase in the expression level of MHC class Ⅱ molecule(25.167%±1.203%)on the surface of immature DCs compared with the control group(P<0.001),and DCM@MBs presented with microbubbles encapsulated by red cell membranes,with uniform dispersion and a size of 1-5 μm.In the in vitro co-culture experiment,the amount of breast cancer cells(9.893±0.341)%.Conclusion:The DCM@MBs strategy proposed in this study shows significant potential in tu-mor immunotherapy and can effectively activate T cells and specifically kill and eliminate tumor cells,which provides new ideas for tu-mor immunotherapy.

Objective:To evaluate the role of signal transducer and activator of transcription 3 (STAT3)/nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in salidroside-induced attenuation of intestinal ischemia-reperfusion (I/R) injury in mice and the relationship with ferroptosis.Methods:Thirty-six SPF-grade healthy male C57BL mice, aged 6-8 weeks, weighing 20-25 g, were divided into 6 groups ( n=6 each) by a random number table method: sham operation group (S group), sham operation+ salidroside group (SS group), intestinal I/R group (IR group), intestinal I/R+ salidroside group (IS group), intestinal I/R+ salidroside+ autophagy activator rapamycin group (ISR group) and intestinal I/R+ salidroside+ STAT3 activator colivelin group (ISC group). The intestinal I/R injury model was established by clamping the superior mesenteric artery for 45 min followed by 30-min reperfusion in IR, IS, ISR and ISC groups, while the superior mesenteric artery was only isolated without clipping in S and SS groups. At 1 week before developing the model, salidroside 40 mg/kg was intraperitoneally injected once a day for 7 consecutive days in SS, IS, ISC and ISR groups, rapamycin 4 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISR, colivelin 1 mg/kg was intraperitoneally injected once a day for 7 consecutive days in group ISC, while the equal volume of normal saline was given instead in the rest two groups. The mice were sacrificed at 30 min of reperfusion, and intestinal tissues were obtained for examination of the pathological changes after HE staining (with a optical microscope) which were scored according to Chiu and for determination of contents of malondialdehyde (MDA), Fe 2+, glutathione (GSH) and reactive oxygen species (ROS), activity of superoxide dismutase (SOD) and expression of p-STAT3, STAT3, glutathione peroxidase 4 (GPX4), NCOA4 and ferritin heavy chain 1 (FTH1) in intestinal tissues (by Western blot). Results:Compared with group S, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly increased, the content of GSH was decreased, the activity of SOD was decreased, the expression of p-STAT3 and NCOA4 was up-regulated, the expression of GPX4 and FTH1 was down-regulated, the p-STAT3/STAT3 ratio was increased ( P<0.05), pathological injury was found in intestinal tissues, and no significant change was found in the aforementioned indexes in group IR( P>0.05). Compared with group IR, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly decreased, GSH content was increased, SOD activity was increased, the expression of p-STAT3 and NCOA4 was down-regulated, the expression of GPX4 and FTH1 was up-regulated, p-STAT3/STAT3 ratio was decreased ( P<0.05), and the pathological injury was significantly alleviated in intestinal tissues in group IS. Compared with group IS, the Chiu′s score and contents of MDA, Fe 2+ and ROS were significantly increased, GSH content was decreased, SOD activity was decreased, the expression of p-STAT3 and NCOA4 was up-regulated, the expression of GPX4 and FTH1 was down-regulated, p-STAT3/STAT3 ratio was increased ( P<0.05), and the pathological injury was aggravated in intestinal tissues in ISR and ISC groups. There was no statistically significant difference in the expression of STAT3 among the five groups ( P>0.05). Conclusions:STAT3/NCOA4-mediated ferritinophagy is involved in the process of salidroside-induced reduction of intestinal I/R injury in mice, which may be related to inhibiting ferroptosis.

Objective:To evaluate the role of endoplasmic reticulum stress-mediated apoptosis in proanthocyanidins-induced attenuation of intestinal ischemia-reperfusion (I/R) injury in mice.Methods:Thirty SPF healthy adult male C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 5 groups ( n=6 each) by a random number table method: sham operation group (S group), sham operation + proanthocyanidins group (S+ PC group), intestinal I/R group (I/R group), intestinal I/R + proanthocyanidins group (I/R+ PC group) and intestinal I/R + proanthocyanidins + tunicamycin group (I/R+ PC+ TM group). The superior mesenteric artery was clamped for 60 min and reperfused for 120 min to establish a mouse intestinal I/R injury model.Proanthocyanidin 100 mg/kg was given by intragastric gavage every day 1 week before ischemia in S+ PC group, I/R+ PC group and I/R+ PC+ TM group, and the equal volume of normal saline was given for 7 consecutive days in S group and I/R group, and endoplasmic reticulum stress agonist tunicamycin 1 mg/kg was intraperitoneally injected at 24 h before ischemia in I/R+ PC+ TM group.The mice were sacrificed at 120 min of reperfusion, and the small intestinal tissues were taken for microscopic examination of the histopathological changes (using light microscopy) and for determination of the level of diamine oxidase (DAO) (by enzyme-linked immunosorbent assay), cell apoptosis (by TUNEL method), glucose regulatory protein 78 (GRP78), C/EBP-homologous protein (CHOP) and cleaved caspase-3, Bax and Bcl-2 (by Western blot). Intestinal damage was assessed and scored according to Chiu, and the apoptosis index (AI) and Bcl-2/Bax ratio were calculated. Results:Compared with S group, the Chiu′s score, level of DAO and AI were significantly increased, the expression of GRP78, CHOP, cleaved caspase-3 and Bax was up-regulated, the expression of Bcl-2 was down-regulated, the ratio of Bcl-2/Bax was decreased( P<0.05), and the pathological damage occurred in the small intestinal tissue in I/R group, and no significant change was found in the aforementioned indexes in S+ PC group ( P>0.05). Compared with I/R group, the Chiu′s score, DAO level and AI were significantly decreased, the expression of GRP78, CHOP, cleaved caspase-3 and Bax was down-regulated, the expression of Bcl-2 was up-regulated, the ratio of Bcl-2/Bax was increased( P<0.05), and the pathological injury to the small intestinal tissue was significantly reduced in I/R+ PC group. Compared with I/R+ PC group, the Chiu′s score, level of DAO and AI were significantly increased, the expression of GRP78, CHOP, cleaved caspase-3 and Bax was up-regulated, the expression of Bcl-2 was down-regulated, and the ratio of Bcl-2/Bax was decreased( P<0.05), and the pathological damage to the small intestinal tissue was aggravated in I/R+ PC+ TM group. Conclusions:Proanthocyanidins can alleviate intestinal I/R injury by inhibiting endoplasmic reticulum stress-mediated cell apoptosis in mice.

Objective:To evaluate the role of silencing information regulatory factor 1/nuclear factor E2 related factor 2 (SIRT1/Nrf2) signaling pathway in berberine preconditioning-induced reduction of intestinal ischemia-reperfusion (I/R) injury in mice and the relationship with ferroptosis.Methods:Thirty-six SPF-grade healthy male C57BL/6 mice, aged 8-10 weeks, weighing 22-25 g, were divided into 6 groups ( n=6 each) by a random number table method: sham operation group (S group), sham operation + berberine preconditioning group (SB group), intestinal I/R group (IR group), intestinal I/R + berberine preconditioning group (B group), intestinal I/R + berberine preconditioning + SIRT1 inhibitor EX527 group (BE group) and berberine preconditioning + intestinal I/R + ferroptosis inducer RSL3 group (BR group). The model of intestinal I/R injury was prepared by clamping the superior mesenteric artery for 45 min followed by 30-min reperfusion in IR group, B group, BE group and BR group, while the superior mesenteric artery was only isolated without ligation in S group and SB group. Berberine 50 mg/kg was administered by intragastric gavage once a day starting from 7 days before developing the model in SB group, B group, BE group and BR group. EX527 5 mg/kg and RSL3 5 mg/kg were intraperitoneally injected once a day at 3 days before surgery in BE group and BR group, respectively. The equal volume of normal saline was given in the other groups. The mice were sacrificed at 30 min of reperfusion, and the intestinal tissues were taken for microscopic examination of the pathological changes of intestinal mucosa (with a light microscope) which was scored according to Chiu and for determination of the contents of Fe 2+ and malondialdehyde (MDA) and superoxide dismutase (SOD) activity (by colorimetry), glutathione (GSH) content (by enzyme-linked immunosorbent assay), reactive oxygen species (ROS) content (by fluorescence staining), and expression of glutathione peroxidase 4 (GPX4), SIRT1 and Nrf2 (by Western blot). Results:Compared with S group, Chiu′s score was significantly increased, the contents of Fe 2+, MDA and ROS were increased, the content of GSH and activity of SOD were decreased, the expression of GPX4 was down-regulated, and the expression of SIRT1 and Nrf2 was up-regulated in IR group ( P<0.05), and no significant change was found in Chiu′s score in SB group ( P>0.05). Compared with IR group, Chiu′s score was significantly decreased, the contents of Fe 2+, MDA and ROS were decreased, the content of GSH and activity of SOD were increased, the expression of GPX4 was up-regulated, and the expression of SIRT1 and Nrf2 was up-regulated in B group ( P<0.05). Compared with B group, Chiu′s score was significantly increased, the contents of Fe 2+, MDA and ROS were increased, the content of GSH and activity of SOD were decreased, and the expression of GPX4 was down-regulated in BE and BR groups, and the expression of SIRT1 and Nrf2 was down-regulated in BE group( P<0.05). Conclusions:The mechanism by which berberine preconditioning reduces intestinal I/R injury may be associated with activation of SIRT1/Nrf2 signaling pathway, thus inhibiting ferroptosis in mice.

Objective:To evaluate the effects of esketamine on myocardial injury and the relationship with nuclear factor-erythroid 2-related factor 2 (Nrf2) heme oxygenase-1 (HO-1) signaling pathway in septic rats.Methods:Thirty-two SPF healthy adult male Sprague-Dawley rats, weighing 200-230 g, were randomized into 4 groups ( n=8 each) using a random number table method: control group (group C), control plus esketamine group (group CE), sepsis group (group S) and sepsis plus esketamine group (group SE). Lipopolysaccharide (LPS) 10 mg/kg was intraperitoneally injected to establish the sepsis model.At 30 min after LPS or normal saline intraperitoneal injection, esketamine 10 mg/kg was intraperitoneally injected, and administration was repeated 12 h later in group SE and group CE.At 24 h after LPS injection, left ventricular ejection fraction (LVEF) was measured (using echocardiography), and serum cardiac troponin I (cTnI), brain natriuretic peptide (BNP), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), tumor necrosis factor alpha (TNF-α) and high mobility group box-1 (HMGB1) concentrations were determined (by enzyme linked immunosorbent assay). Myocardial tissues were obtained for examination of pathological changes (by hematoxylin-eosin staining) and for determination of expression of Nrf2, HO-1 and transcription factor Bach 1 (BTB-CNC allogeneic 1). Results:Compared with group C, LVEF was significantly decreased, concentrations of cTnI, BNP, LDH, CK-MB, TNF-α and HMGB1 in serum were increased, expression of Nrf2 and HO-1 was down-regulated, and expression of Bach 1 was up-regulated ( P<0.05), and the significant pathological changes of myocardial tissues were found in S and SE groups.No significant change was found in the parameters mentioned above in group CE ( P>0.05). Compared with group S, LVEF was significantly increased, concentrations of cTnI, BNP, LDH, CK-MB, TNF-α and HMGB1 in serum were decreased, expression of Nrf2 and HO-1 was up-regulated, and expression of Bach 1 was down-regulated ( P<0.05), and the pathological changes of myocardium were significantly attenuated in group SE. Conclusion:Esketamine can reduce myocardial injury, and the mechanism may be related to activating Nrf2/HO-1 signaling pathway in septic rats.

Objective: To evaluate the effect of penehyelidine hydrochloride (PHCD) on tumor necrosis factor α-induced protein 8-like-2 (TIPE2)-Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88) signaling pathway in a rat model of traumatic acute lung injury (ALI). Methods: Thirty SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 190-210 g, were divided into 3 groups (n=15 each) by a random number table method: sham operation group (group Sham), traumatic ALI group (group ALI) and group PHCD.ALI was induced by blunt chest trauma in ALI and PHCD groups.PHCD 2 mg/kg was intraperitoneally injected immediately after blunt chest trauma in group PHCD.The rats were sacrificed and lung tissues were removed at 8 h after the model was successfully established for examination of the pathological changes and ultrastructure of lung tissues (with a light microscope or an electron microscope) and for determination of the wet to dry weight ratio (W/D ratio) and expression of TLR4 and MyD88 in lung tissues. Results: Compared with group Sham, the W/D ratio was significantly increased, TIPE2 expression was down-regulated, and the expression of TLR4 and MyD88 was up-regulated in ALI and PHCD groups (P<0.05). Compared with group ALI, the W/D ratio was significantly decreased, TIPE2 expression was up-regulated, and the expression of TLR4 and MyD88 was down-regulated (P<0.05), and the pathological changes of lung tissues and ultrastructure were significantly attenuated in group PHCD. Conclusion The mechanism by which PHCD reduces traumatic AIL is related to activating TIPE2-TLR4-MyD88 signaling pathway in rats.

Objective: To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) and caspase-11 during pyroptosis in macrophages of mice. Methods: J774A.1 macrophages of mice were divided into 4 groups (n=6 each) using a random number table method: non-specific siRNA (Scr-siRNA) group (S group), Scr-siRNA plus LPS/ATP group (S+ LPS/ATP group), TIPE2-siRNA group (T group) and TIPE2-siRNA plus LPS/ATP group (T+ LPS/ATP group). The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group, and transfection was performed for 24-48 h, and in addition LPS 1.0 μg/ml was then added, cells were incubated for 5 h, then ATP 5.0 mmol/L was added, and cells were incubated for 1 h in S+ LPS/ATP and T+ LPS/ATP groups.Cells were collected to detect the expression of TIPE2, caspase-11, NOD-like receptor familypyrin domain containing 3 (NLRP3) and caspase-1 (by Western blot). The supernatant was collected for determination of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) activities and concentrations of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results: Compared with group S, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group S+ LPS/ATP (P<0.05). Compared with group T, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Compared with group S+ LPS/ATP, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Conclusion TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice.

Objective To evaluate the effect of penehyelidine hydrochloride(PHCD)on tumor necrosis factor α-induced protein 8-like-2(TIPE2)-Toll-like receptor 4(TLR4)-myeloid differentiation fac-tor 88(MyD88)signaling pathway in a rat model of traumatic acute lung injury(ALI).Methods Thirty SPF healthy male Sprague-Dawley rats,aged 8 weeks,weighing 190-210 g,were divided into 3 groups(n＝15 each)by a random number table method: sham operation group(group Sham),traumatic ALI group(group ALI)and group PHCD.ALI was induced by blunt chest trauma in ALI and PHCD groups.PHCD 2 mg/kg was intraperitoneally injected immediately after blunt chest trauma in group PHCD.The rats were sacrificed and lung tissues were removed at 8 h after the model was successfully established for exami-nation of the pathological changes and ultrastructure of lung tissues(with a light microscope or an electron microscope)and for determination of the wet to dry weight ratio(W/D ratio)and expression of TLR4 and MyD88 in lung tissues.Results Compared with group Sham,the W/D ratio was significantly increased,TIPE2 expression was down-regulated,and the expression of TLR4 and MyD88 was up-regulated in ALI and PHCD groups(P＜0.05).Compared with group ALI,the W/D ratio was significantly decreased,TIPE2 expression was up-regulated,and the expression of TLR4 and MyD88 was down-regulated(P＜0.05),and the pathological changes of lung tissues and ultrastructure were significantly attenuated in group PHCD.Conclusion The mechanism by which PHCD reduces traumatic AIL is related to activating TIPE2-TLR4-MyD88 signaling pathway in rats.

Objective To evaluate the effects of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway on the brain injury induced by myocardial ischemia-reperfusion (I/R) in diabetic rats.Methods Pathogen-free male Sprague-Dawley rats,weighing 200-220 g,were used in this study.Diabetes mellitus was induced by intraperitoneal 1％ streptozotocin 60 mg/kg and confirmed by blood glucose level ≥ 16.7 mmol/L 3 days later.Twenty-four rats with diabetes mellitus were randomly allocated into 3 groups (n =8 each) using a random number table:sham operation group (group S),I/R group,and myocardial I/R + AG490 (JAK inhibitor) group (group ⅠA).Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min,followed by 120 min of reperfusion in the rats anesthetized with pentobarbital sodium.AG490 3 mg/kg was injected intravenously at 20 min before reperfusion in group IA.The rats were sacrificed at 120 min of reperfusion,and the brains were removed for determination of caspase-3 and nuclear factor kappa B (NF-κB) activities (using colorimetric method),cell apoptosis (by TUNEL),and expression of interleukin-1 (IL-1),IL-6,IL-8,Bax,Bcl-2,cytochrome C (Cyt c),phosphorylated JAK2 (p-JAK2),and phosphorylated STAT3 (p-STAT3) (by Western blot).Apoptosis index was calculated.Results Compared with group S,the expression of Bax,Cyt c,IL-1,IL-6,IL-8,p-JAK2 and p-STAT3 was significantly up-regulated,the expression of Bcl-2 was down-regulated,and NF-κB and caspase-3 activities and apoptosis index were increased in I/R and IA groups (P＜0.05).Compared with group I/R,the expression of Bax,Cyt c,IL-1,IL-6,IL-8,p-JAK2 and p-STAT3 was significantly down-regulated,the expression of Bcl-2 was up-regulated,and NF-κB and caspase-3 activities and apoptosis index were decreased in group IA (P＜0.05).Conclusion Inflammatory responses mediated by JAK2/STAT3 signaling pathway are involved in the brain injury induced by myocardial I/R in diabetic rats.

Objective To investigate the effects of penehyclidine hydrochloride on activities of nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) during actue lung injury induced by blunt chest trauma-hemorrhagic shock and resuscitation (HSR) in rats.Methods Thirty male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly assigned into 3 equal groups (n =10 each) using a random number table:sham operation group (group S),blunt chest trauma-HSR group (group THSR) and penehyclidine hydrochloride group (group PHCD).The model of actue lung injury induced by blunt chest trauma-HSR was induced by dropping a 300 g weight onto a precordium in anesthetized rats.Blood was withdrawn via the femoral artery 5 min later until MAP was decreased to 35-45 mmHg within 15 min and maintained at this level for 60 min,followed by resuscitation.In PHCD group,PHCD 2 mg/kg was injected intravenously at 60 min after hemorrhagic shock.At 6 h after the model was established,blood samples were obtained for measurement of concentrations of tumor necrosis factor-alpha (TNF-α) in serum.The lungs were then removed for determination of lung water content,myeloperoxidase (MPO) activaty (by colorimetric assay),NF-κB and AP-1 activaties (using electrophoretic mobility shift assay) in lung tissues,and for microscopic examination of pathologic changes (under light microscope).The left lung was lavaged,and lung permeability index (LPI) was calculated.Results Compared with S group,lung water content,LPI,serum TNF-α level and activites of MPO,NF-κB and AP-1 were significantly increased in THSR and PHCD groups.Compared with THSR group,lung water content,LPI,serum TNF-α concentrations and activites of MPO,NF-κB and AP-1 were significantly decreased in PHCD group.The pathological damage to lung tissues was significantly reduced in PHCD group as compared with THSR group.Conclusion PHCD can inhibit activities of NF-κB and AP-1 in lung tissues,thus mitigating acute lung injury induced by blunt chest trauma-HSR in rats.