Effect of Cistanches Herba Phenylethanoid Glycosides on Intestinal Mucosal Barrier and Gut Microbiota in ALD Mice / 中国实验方剂学杂志

ObjectiveTo discuss the effects of Cistanches Herba phenylethanoid glycosides （CHPhGs） on the intestinal mucosal barrier and gut microbiota in alcoholic liver disease （ALD） mice were discussed. MethodThe 36 C57BL/6N female mice were randomly divided normal group， normal group of CHPhGs， model group， and low， medium， and high-dose groups （175， 350， 700 mg·kg-1） of CHPhGs， with six mice in each group. The ALD mouse model was built using Lieber-Decarli alcohol liquid feed. The normal group and low， medium， and high-dose groups of CHPhGs were given CHPhGs by gavage daily. Serum aspartate aminotransferase aminotransferase （ALT）， alanine aminotransferase （AST）， triglycerides （TG）， and total cholesterol （TC） levels were detected by an automatic biochemical analyzer. Serum tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， lipopolysaccharide （LPS）， lipopolysaccharide-binding protein （LBP）， D-lactic acid （D-LA）， diamine oxidase （DAO）， and LBP of liver were detected by enzyme-linked immunosorbent assay （ELISA）. The levels of TG and TC in the liver were detected by colorimetry. Liver tissue was treated by oil red O and hematoxylin-eosin （HE） staining. The microstructure of jejunum epithelial cells was observed by electron microscope. Jejunum and colon were treated by HE staining and alcian blue-periodate-scheff (AB-PAS) staining staining， and mucin 2 （Muc2） was treated by immunohistochemistry. The intestinal contents of the normal group， normal group of CHPhGs， model group， and high-dose group of CHPhGs were collected and sequenced. ResultThe ALD model was established successfully. Compared with the normal group， the levels of serum ALT， AST， and TG， as well as the levels of liver TG and TC in the model group were significantly increased （P<0.05）. Histopathology showed that compared with the normal group， the liver cells in the model group showed obvious steatosis. Compared with the model group， the levels of serum TG and liver TG and TC in the low， medium， and high-dose groups of CHPhGs decreased significantly （P<0.05）. The serum ALT， AST， TNF-α， IL-1β， LPS， and LBP in the high-dose group of CHPhGs were also significantly decreased （P<0.05）. The number of liver cells with steatosis in the high-dose group of CHPhGs was significantly reduced， and the microvilli structure of jejunum epithelial cells was basically intact. The expression of Muc2 was reduced in the colon， and the gut microbiota of the high-dose group of CHPhGs changed significantly （P<0.05）. Compared with the normal group， the Allobaculum was significantly up-regulated in the model group （P<0.05）. Compared with the model group， the abundance of Akkermansia in the high-dose group of CHPhGs was significantly increased （P<0.01）. The abundance of Akkermansia was negatively correlated with that of Allobaculum （r=-0.701， P<0.01）. ConclusionCHPhGs can reduce the intestinal barrier injury caused by ALD， which may play a protective role by regulating the abundance and structure of Akkermansia and Allobaculum and affecting the homeostasis of intestinal mucus.

Three-dimensional magnetization prepared rapid acquisition gradient echo for evaluation on the corpus callosum morphological alterations in children with spastic cerebral palsy / 实用放射学杂志

Objective To analyze the morphological alterations of corpus callosum in children with spastic cerebral palsy(SCP)using three-dimensional magnetization prepared rapid acquisition gradient echo(3D-MPRAGE)technology and to investigate the correlation between morphological indexes and gross motor function.Methods Sagittal T1WI 3D-MPRAGE data was collected from 136 children with SCP(SCP group)and 132 age-and gender-matched healthy controls(HC)(HC group),and the gross motor function measure-88(GMFM-88)was applied to assess the gross motor function.Independent sample t-test was used to compare the corpus callosum surface area,volume,maximum anterior-posterior diameter,median sagittal area(total area and area of Ⅰ-Ⅴ zone)between the two groups.Partial correlation analysis was performed to calculate the correlation between morphological indexes of the corpus callosum and GMFM-88 with age as a covariate.Results Children under 3 years old,the corpus callosum surface area of the SCP group(3 914.51 mm2±1 207.97 mm2)was lower than that of the HC group(5 725.51 mm2±1 412.66 mm2).The volume of the corpus callosum(6 108.46 mm3±2 803.97 mm3)in the SCP group was lower than that of the HC group(11 297.96 mm3±4 109.02 mm3).Also,the maximum anterior-posterior diameter of the corpus callosum in the SCP group(53.40 mm±6.31 mm)was lower than that of the HC group(57.74 mm±6.04 mm)(all P<0.05).Children over 3 years old,the corpus callosum surface area of the SCP group(4 970.06 mm2±1 191.31 mm2)was lower than that of the HC group(6 372.55 mm2±1 445.59 mm2).The volume of the corpus callosum(8 330.20 mm3±2 888.20 mm3)in the SCP group was lower than that of the HC group(13 599.82 mm3±3 429.81 mm3)(all P<0.05).Partial correlation analysis showed significant correlation between corpus callosum volume,median sagittal area and gross motor score(P<0.01)with age as a covari-ate.Conclusion The 3D-MPRAGE technology can be useful for the comprehensive assessment of morphological alterations of the corpus callosum in SCP.The corpus callosum volume,and median sagittal area may become neuroimaging references for the assess-ment of motor development in cerebral palsy(CP).

Clinical and imaging analysis of neurological complications in critically ill children infected with SARS-CoV-2 Omicron / 中华危重病急救医学

OBJECTIVE@#To summarize clinical predictors and imaging characteristics of critically ill children infected with SARS-CoV-2 Omicron with neurological complications in Shenzhen during the peak of the first round of infections.@*METHODS@#The clinical data of 11 critically ill children with neurological complications infected with SARS-CoV-2 Omicron in Shenzhen Children's Hospital from December 12 to 31, 2022, were retrospectively collected and analyzed. Laboratory test results related to liver parenchymal injury, histiocytic injury, inflammation, and coagulation function were collected, and imaging characteristics including CT and/or magnetic resonance imaging (MRI) were analyzed. The differences in CT/MRI score, acute necrotizing encephalopathy severity scale (ANE-SS) score and total score (CT/MRI score + ANE-SS score) were compared between the two groups with different prognosis during hospitation.@*RESULTS@#Among 11 children, 7 were male and 4 were female. The age ranged from 10 months to 16 years. There were 5 cases of acute necrotizing encephalopathy (ANE) and 6 cases of acute fulminant cerebral edema (AFCE). During hospitalization, 3 patients survived and 8 patients died of multiple organ dysfunction syndrome (MODS), including 2 cases of ANE and 6 cases of AFCE. All cases had fever (> 38.5 centigrade), and 3 cases had ultra-high fever (> 41 centigrade). Within 48 hours of onset, all cases had disorders of consciousness and 9 cases had seizures. The 8 dead children had complications with multisystem involvement, including shock, respiratory failure, disseminated intravascular coagulation (DIC), liver failure, renal failure or myocardial damage, and the laboratory predictors related to hepatocellular injury [alanine aminotransferase (ALT), aspartate aminotransferase (AST)], histocyte injury [creatine kinase (CK), lactate dehydrogenase (LDH)], inflammation [procalcitonin (PCT), interleukin-6 (IL-6), serum ferritin (SF)], coagulation function (D-dimer) and blood glucose (Glu) increased in different quantities, of which PCT was specifically increased in 6 cases with AFCE, PLT was specifically decreased in 3 cases with AFCE, and ALT and LDH were significantly increased in 2 cases with ANE. Imaging analysis showed subarachnoid hemorrhage, basal ganglia and thalamus lesions in all 6 cases with AFCE, while thalamus lesions in all 5 cases with ANE. The ANE-SS score of 8 deceased children ranged from 2 to 7 (of which 6 cases were ≥ 5), and the ANE-SS score of 3 surviving children ranged from 0 to 2. Eight dead children had a CT/MRI score of 1-4 (of which 6 cases were 4), and 3 surviving children had a CT/MRI score of 1-2 (of which 2 cases were 1). The total score of 8 deceased children was 6-10 (of which 6 cases ≥ 8), and 3 surviving children was 1-4.@*CONCLUSIONS@#The neurological complications of critically ill children infected with SARS-CoV-2 Omicron in Shenzhen progressed rapidly to ANE and AFCE, with high mortality. High fever (> 40 centigrade), convulsion/disturbance of consciousness, and multiple organ failure were the most common symptoms in ANE and AFCE cases. PCT increased and PLT decreased specifically in AFCE cases. Poor prognosis (death) was more common in age < 4 years old, predictors of ALT, AST, CK, LDH, PCT, D-dimer, Glu, IL-6 increased significantly, PLT decreased significantly. The common imaging feature of ANE and AFCE is the involvement of dorsal thalamus, a new imaging sign of AFCE (subarachnoid hemorrhage) was found. The higher the ANE-SS score, CT/MRI score and total score, the greater the risk of death.

Mechanism of heparin-binding protein increasing vascular permeability in early burn / 中华危重病急救医学

Objective:To investigate the effect of heparin-binding protein (HBP) on the damage of vascular permeability in early burn.Methods:① Clinical research: 12 patients with severe burns admitted to Suzhou Hospital of Nanjing Medical University from January 1st to August 30th in 2019 were enrolled. Eight patients with severe trauma admitted to the same hospital during the same period were also enrolled as controls to explain the specificity of burn injury. Whole blood samples were obtained within 0.5 hour after admission. The white blood cell count (WBC), absolute value and ratio of neutrophils, and serum HBP levels were measured. Serum samples of 12 patients with severe burn were collected within 9 days after admission, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of metabolism products of glycocalyx including syndecan-1 and hyaluronic acid (HA). The correlation between HBP and neutrophils ratio, syndecan-1 and HA were analyzed by linear correlation. ② Basic research: a 30% total body surface area (TBSA) Ⅲ° burn model of Sprague-Dawley (SD) rat aged 6-8 weeks was prepared. In low molecular weight heparin (LMWH) intervention group ( n = 5), 200 U/kg LMWH was injected subcutaneously immediately and every 2 hours after injury for 4 times in total; the burn group ( n = 5) was given the same amount of normal saline. No intervention was given to the normal control group ( n = 5). The peripheral venous blood was collected at 0, 2, 4, and 8 hours after injury, and the serum levels of HBP, syndecan-1 and HA were measured; the injury of glycocalyx on pulmonary vascular endothelial cells was observed under transmission electron microscope. Results:① Clinical research results: the WBC, neutrophils absolute value and ratio, and HBP levels were increased in 12 patients with severe burn and 8 patients with severe trauma. There was no significant difference in the WBC, absolute value and ratio of neutrophils between severe burn and severe trauma patients [WBC (×10 9/L): 14.5±6.1 vs. 10.8±3.6, the absolute value of neutrophils (×10 9/L): 12.0±5.9 vs. 9.0±4.0, the ratio of neutrophils: 0.81±0.10 vs. 0.79±0.14, all P > 0.05], but the HBP levels in the burn patients were significantly higher than those in the trauma patients (μg/L: 192.92±61.73 vs. 51.17±23.05, P < 0.01). Twelve patients with severe burns had a sharp increase in serum syndecan-1 and HA levels after burns, which continued to maintain high levels and peaked at the 9th day [syndecan-1 (μg/L): 16.02±0.39, HA (μg/L): 106.83±4.90]. The analysis showed that HBP was positively correlated with neutrophils ratio, syndecan-1 and HA in severe burn patients at the 1st day after admission ( r values were 0.805, 0.732 and 0.900, respectively, all P < 0.01). It indicated that the sharp increase of neutrophils after the burn released a lot of HBP, and the glycocalyx of the vascular endothelium was severely damaged. ② Basic research results: the levels of serum HBP, syndecan-1 and HA in the burn group were increased sharply as compared with the normal control group, and continued to increase with time, reaching a peak at 8 hours after burn. In the LMWH intervention group, the serum levels of HBP, syndecan-1 and HA were significantly lower than those in the burn group, and the difference was still statistically significant after 8 hours [HBP (μg/L): 6.47±0.25 vs. 12.48±0.08, syndecan-1 (μg/L): 19.06±1.48 vs. 25.92±3.34, HA (μg/L): 35.76±2.10 vs. 54.91±2.64, all P < 0.01]. The results of transmission electron microscopy showed that in the normal control group, the glycocalyx pulmonary vascular endothelial cells was continuous, evenly distributed and dense. The glycocalyx on pulmonary vascular endothelial cells of rats were significantly damaged and shed 2 hours after burn in the burn group, and no glycocalyx was observed at 8 hours. In the LMWH intervention group, the glycocalyx on pulmonary vascular endothelial cells was damaged and the phenomenon of shedding was significantly relieved, and the glycocalyx could be observed 8 hours after the injury. Conclusion:The massive exudation of body fluids and the significant increase of vascular permeability in patients in early burns may be related to the destruction of the glycocalyx on endothelial cells by HBP released from increased neutrophils.

Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis / 中华危重病急救医学

Objective To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis. Methods ① In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg·kg-1·d-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. ② In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot. Results① In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice.② In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth. Conclusions In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.

Mechanism of delayed apoptosis of peripheral blood neutrophils in severely burned patients / 中华危重病急救医学

Objective To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients. Methods Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep Ⅱ to Ⅲ degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot. Results Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05). Conclusion Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

Mechanism of delayed apoptosis of peripheral blood neutrophils in severely burned patients / 中华危重病急救医学

Objective To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients. Methods Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep Ⅱ to Ⅲ degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot. Results Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05). Conclusion Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

Mechanism of delayed apoptosis of peripheral blood neutrophils in severely burned patients / 中华危重病急救医学

Objective@#To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients.@*Methods@#Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep Ⅱ to Ⅲ degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot.@*Results@#Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05).@*Conclusion@#Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis / 中华危重病急救医学

Objective@#To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis.@*Methods@#① In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg·kg-1·d-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. ② In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot.@*Results@#① In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. ② In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth.@*Conclusions@#In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.

Risk factors for cognitive impairment after ischemic stroke / 国际脑血管病杂志

Early diagnosis and early intervention for cognitive impairment after ischemic stroke can delay disease progression and prevent the occurrence of dementia.This article reviews the advances in research on ischemic stroke caused each risk factor for cognitive impairment.