BACKGROUND:In vitro construction of tissue-engineered intestinal models plays an important role in intestinal regeneration and intestinal disease research.The interaction of intestinal nervous system and intestinal epithelial barrier to maintain body homeostasis is a hot topic in the bionic construction of tissue-engineered intestinal tract.OBJECTIVE:To construct a bionic model that can mimic the enteric nervous system in vivo.METHODS:Using fibroin protein with villus structure as scaffold,human induced neural stem cells solidified with collagen were added to intestinal epithelial cells(Caco-2 and HT29-MTX-E12)for 3-day culture to construct a co-culture system of intestinal epithelial cells and nerve cells(co-culture group).Human induced neural stem cells or intestinal epithelial cells cultured alone that were inoculated with fibroin scaffolds were set as controls.Cell morphology was observed by scanning electron microscopy and hematoxylin-eosin staining.Cell activity was detected by Live/Dead cell staining.Human induced neural stem cell differentiation was detected by β-microtubulin immunofluorescence staining.Intestinal epithelial histological properties and barrier function were detected by microvillin,sucrase-isomaltase,tight junction protein 1,E-calmodulin,and mucin-2 immunofluorescence staining.The function of mucus secretion from intestinal epithelial cells was detected by Alcian blue staining.Alkaline phosphatase staining was performed to detect differentiation of intestinal epithelial cells,at the same time,sucrase-isomaltase,tight junction protein 1,and alkaline phosphatase mRNAs were detected by RT-qRCR.RESULTS AND CONCLUSION:The neuralized intestinal mucosal co-culture model with villi structure was successfully constructed,and neural stem cells and intestinal epithelial cells on the fibroin scaffold showed good cellular activities.After neuralization,the activity of alkaline phosphatase and sucrase-isomaltase in intestinal epithelial cells was enhanced,while the expression level of tight junction protein 1 was up-regulated.To conclude,the neuralized bionic intestinal epithelial model is beneficial to the maturation of intestinal mucosal epithelial cells and the formation of barrier function.

BACKGROUND:In vitro construction of tissue-engineered intestinal models plays an important role in intestinal regeneration and intestinal disease research.The interaction of intestinal nervous system and intestinal epithelial barrier to maintain body homeostasis is a hot topic in the bionic construction of tissue-engineered intestinal tract.OBJECTIVE:To construct a bionic model that can mimic the enteric nervous system in vivo.METHODS:Using fibroin protein with villus structure as scaffold,human induced neural stem cells solidified with collagen were added to intestinal epithelial cells(Caco-2 and HT29-MTX-E12)for 3-day culture to construct a co-culture system of intestinal epithelial cells and nerve cells(co-culture group).Human induced neural stem cells or intestinal epithelial cells cultured alone that were inoculated with fibroin scaffolds were set as controls.Cell morphology was observed by scanning electron microscopy and hematoxylin-eosin staining.Cell activity was detected by Live/Dead cell staining.Human induced neural stem cell differentiation was detected by β-microtubulin immunofluorescence staining.Intestinal epithelial histological properties and barrier function were detected by microvillin,sucrase-isomaltase,tight junction protein 1,E-calmodulin,and mucin-2 immunofluorescence staining.The function of mucus secretion from intestinal epithelial cells was detected by Alcian blue staining.Alkaline phosphatase staining was performed to detect differentiation of intestinal epithelial cells,at the same time,sucrase-isomaltase,tight junction protein 1,and alkaline phosphatase mRNAs were detected by RT-qRCR.RESULTS AND CONCLUSION:The neuralized intestinal mucosal co-culture model with villi structure was successfully constructed,and neural stem cells and intestinal epithelial cells on the fibroin scaffold showed good cellular activities.After neuralization,the activity of alkaline phosphatase and sucrase-isomaltase in intestinal epithelial cells was enhanced,while the expression level of tight junction protein 1 was up-regulated.To conclude,the neuralized bionic intestinal epithelial model is beneficial to the maturation of intestinal mucosal epithelial cells and the formation of barrier function.

Background: Intraoperative hypercapnia reduces the time to emergence from volatile anesthetics, but few clinical studies have explored the effect of hypercapnia on the emergence time from intravenous (IV) anesthesia. We investigated the effect of inducing mild hypercapnia during the recovery period on the emergence time after total IV anesthesia (TIVA). Methods: Adult patients undergoing transurethral lithotripsy under TIVA were randomly allocated to normocapnia group (end-tidal carbon dioxide [ETCO2] 35–40 mmHg) or mild hypercapnia group (ETCO2 50-55 mmHg) during the recovery period. The primary outcome was the extubation time. The spontaneous breathing-onset time, voluntary eye-opening time, and hemodynamic data were collected. Changes in the cerebral blood flow velocity in the middle cerebral artery were assessed using transcranial Doppler ultrasound. Results: In total, 164 patients completed the study. The extubation time was significantly shorter in the mild hypercapnia (13.9 ± 5.9 min, P = 0.024) than in the normocapnia group (16.3 ± 7.6 min). A similar reduction was observed in spontaneous breathing-onset time (P = 0.021) and voluntary eye-opening time (P = 0.008). Multiple linear regression analysis revealed that the adjusted ETCO2 level was a negative predictor of extubation time. Middle cerebral artery blood flow velocity was significantly increased after ETCO2 adjustment for mild hypercapnia, which rapidly returned to baseline, without any adverse reactions, within 20 min after extubation. Conclusions Mild hypercapnia during the recovery period significantly reduces the extubation time after TIVA. Increased ETCO2 levels can potentially enhance rapid recovery from IV anesthesia.

Objective To investigate the neuroprotective effect of ligustilide(LIG)-mediated phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1)/Parkin pathway on mitophagy in rats with cerebral ischemia-reperfusion injury.Methods 161 male Sprague Dawley(SD)rats were randomly divided into sham operation(Sham)group,model group,LIG low-dose group,LIG high-dose group,mitophagy inhibitor(Mdivi-1)group,LIG high-dose+Mdivi-1 group,and the positive drug Nimodipine(NMDP)group,each with 23 rats.A modified middle cerebral artery wire thrombus method was used to construct a cerebral ischemia/reperfusion model in rats,and the neurobehavioral scores of rats in each group were compared by Longa's five-point scale;the volume of cerebral infarction was detected by 2,3,5-triphenyltetrazolium chloride(TCC)staining,the histopathology and ultrastructure of the hippocampus were examined by hematoxylin-eosin(HE)staining and transmission electron microscope(TEM).And the Na+-K+-Adenosine Triphosphate was measured by enzyme-linked immunosorbent assay(ELISA);double immunofluorescence staining for translocase of the outer membrane of mitochondrion 20(TOMM20)and Microtubule-associated protein 1 light chain 3(LC3)co-localized area percentage.Flow cytometry assay(FCM)to test the level of reactive oxygen(ROS);real-time fluorescence quantitative PCR(qRT-PCR)was used to measure the relative content of mitochondria in hippocampal neurons;and Western blot was performed to test the level of autophagy and the PINK1/Parkin pathway related protein expression.Results Compared with the Sham group,the neurological function score and cerebral infarction volume of the model group were increased,the hippocampal neurons showed pathological damage such as disordered arrangement,nucleolus disappearance and partial shrinkage of the nucleus and plasma,nuclear membrane rupture,swelling,membrane rupture and crista reduction of some mitochondria,a large number of autophagosomes were observed,and the colocalization area percentage of TOMM20 and LC3 was increased.TOMM20 and cytochrome C oxidase subunit IV isoform 1(COX4I1)in hippocampus and selective autophagy adaptor protein 62(p62)protein expression,mitochondrial encoded ATP synthase 6(mt-ATP6)/Ribosomal protein L13(Rpl13)ratio and Na+-K+-ATPase content decreased,while PINK1 and Parkin protein expression,LC3-II/I ratio and ROS relative content increased,and the differences were statistically significant(t=4.602～52.012,all P<0.01).Compared with the model group,the neurological function score,cerebral infarction volume,pathological and ultrastructural damage of hippocampal neurons were significantly improved in the LIG low,high dose and NMDP groups,and the differences were statistically significant(t=4.851～12.525,all P<0.01).The colocalization of TOMM20 and LC3 and the content of Na+-K+-ATPase were increased,while the expression of TOMM20,COX4I1 and p62 proteins and the mt-ATP6/Rpl13 ratio were decreased in the high-dose LIG group.The protein expression of PINK1 and Parkin,LC3-II/I ratio and ROS relative content were increased,and the differences were statistically significant(t=4.087～33.211,all P<0.01).Compared with the LIG high-dose group,the Mdivi-1 and LIG+Mdivi-1 groups had significantly decreased colocalization of TOMM20 and LC3 and Na+-K+-ATPase content,and significantly increased expression of TOMM20,COX4I1 and p62 proteins and mt-ATP6/Rpl13 ratio.The protein expression of PINK1 and Parkin,LC3-II/I ratio and ROS relative content were decreased,and the differences were statistically significant(t=4.008～43.415,all P<0.01).However,the percentage of TOMM20 and LC3 co-localization area,PINK1 and Parkin protein expression,LC3-II/I ratio and Na+-K+-ATPase content in the hippocampus of the LIG+Mdivi-1 group were higher than those of the Mdivi-1 group.The protein expression of COX4I1 and p62,mt-ATP6/Rpl13 ratio and ROS level were lower than those in MDIV-1 group,and the differences were statistically significant(t=3.721～21.513,all P<0.01).Conclusion LIG may activate mitophagy by regulating PINK1/Parkin signaling pathway to protect neurons from cerebral ischemia-reperfusion injury in rats.

BACKGROUND:When exercising in a cold environment,the body's inflammatory response is affected by both low temperature and exercise intervention,and its impact and mechanism remain to be explored.OBJECTIVE:To explore the effects and mechanisms of cold water swimming on inflammatory response of rats based on transcriptome sequencing technology.METHODS:40 male SD rats were randomly divided into room temperature control group,room temperature swimming group,cold water control group,and cold water swimming group,with 10 rats in each group.The room temperature control group had no intervention and was free to eat.The room temperature swimming group received swimming at 30 min/time,6 times/week,for 5 weeks;the water temperature was(28±2)℃,and the water depth was 35 cm.In the cold water control group,the rats were placed in a water tank with a depth of 3 cm;the water temperature was(18±2)℃,and they were free to move.The cold water swimming group received swimming at 30 min/time,6 times/week,for 5 weeks;the water temperature was(18±2)℃,and the water depth was 35 cm.Enzyme-linked immunosorbent assay was used to detect the levels of serum interleukin-6,tumor necrosis factor-α,and high-sensitivity C-reactive protein.Based on the transcriptome sequencing results,differentially expressed genes were screened to draw Venn diagrams and heat maps,and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed.The protein-protein interaction network was used to screen core genes.RT-qPCR was used to detect the mRNA expression of IRF7,OAS2,and OASL in rat spleen tissue.RESULTS AND CONCLUSION:(1)The ELISA results showed that compared with the room temperature control group,the levels of various inflammatory indicators in the room temperature swimming group and the cold water swimming group were significantly increased(P＜0.05),and there was no significant difference in the cold water control group.Compared with the room temperature swimming group,there was no significant difference in the expression of inflammatory indicators in the cold water swimming group.Compared with the cold water control group,the expressions of interleukin-6 and tumor necrosis factor-α in the cold water swimming group showed an upward trend,and high-sensitivity C-reactive protein increased significantly(P＜0.05).(2)Transcriptome analysis:Venn diagram showed that there were 39 differentially expressed genes affected by the dual factors of temperature and exercise intervention.Cluster heat map analysis results showed that the overall gene expression trends of the room temperature swimming group and the cold water swimming group were similar,and the cold water control group showed an opposite trend.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis results showed that differentially expressed genes were enriched in the immune system,locomotion,nucleic acid-binding transcription factor activity,NOD-like receptor signaling pathways and other pathways.The number of genes enriched in the NOD-like receptor signaling pathway was relatively large,and the q value was small,which may be a key pathway.The protein-protein interaction network screened out IRF7,OAS2,OASL,IFIT2,IFIT3 and other core genes.(3)RT-qPCR verification results showed that compared with the room temperature control group,the expressions of IRF7,OAS2 and OASL were significantly increased in the room temperature swimming group and the cold water swimming group(P＜0.01),and there was no significant difference in the cold water control group.Compared with the cold water control group,the expression of each gene was significantly increased in the cold water swimming group(P＜0.01).(4)It is concluded that cold water swimming can promote inflammatory response,and its mechanism may be regulated through the NOD-like receptor signaling pathway.

Freeze-drying technique,also known as lyophilization,is a process of removing moisture from a solution or suspension through freezing and vacuum dehydration to maintain the stability of the samples and prolong their shelf life.Freeze-drying technology has been widely used in food,pharmaceutical,clinical testing,chemical and other fields but its application in the field of forensic medicine has just started.Polymerase chain reaction(PCR)and its derivative detection technologies are widely used in forensic DNA detection,but PCR reagents need to be stored and transported at low temperature.In recent years,forensic scientists have begun applying freeze-drying technology to PCR amplification reagents to solve the transportation and storage problems of PCR reagents.In order to promote the application of PCR freeze-drying technology in forensic genetics,this paper mainly expounds the research course,system and process of PCR freeze-drying technology,compares the advantages and disadvantages of PCR reagents with traditional PCR reagents,and introduces the advantages and challenges of PCR freeze-drying reagents in forensic medicine.

Objective To investigate the neuroprotective effect of ligustilide(LIG)-mediated phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1)/Parkin pathway on mitophagy in rats with cerebral ischemia-reperfusion injury.Methods 161 male Sprague Dawley(SD)rats were randomly divided into sham operation(Sham)group,model group,LIG low-dose group,LIG high-dose group,mitophagy inhibitor(Mdivi-1)group,LIG high-dose+Mdivi-1 group,and the positive drug Nimodipine(NMDP)group,each with 23 rats.A modified middle cerebral artery wire thrombus method was used to construct a cerebral ischemia/reperfusion model in rats,and the neurobehavioral scores of rats in each group were compared by Longa's five-point scale;the volume of cerebral infarction was detected by 2,3,5-triphenyltetrazolium chloride(TCC)staining,the histopathology and ultrastructure of the hippocampus were examined by hematoxylin-eosin(HE)staining and transmission electron microscope(TEM).And the Na+-K+-Adenosine Triphosphate was measured by enzyme-linked immunosorbent assay(ELISA);double immunofluorescence staining for translocase of the outer membrane of mitochondrion 20(TOMM20)and Microtubule-associated protein 1 light chain 3(LC3)co-localized area percentage.Flow cytometry assay(FCM)to test the level of reactive oxygen(ROS);real-time fluorescence quantitative PCR(qRT-PCR)was used to measure the relative content of mitochondria in hippocampal neurons;and Western blot was performed to test the level of autophagy and the PINK1/Parkin pathway related protein expression.Results Compared with the Sham group,the neurological function score and cerebral infarction volume of the model group were increased,the hippocampal neurons showed pathological damage such as disordered arrangement,nucleolus disappearance and partial shrinkage of the nucleus and plasma,nuclear membrane rupture,swelling,membrane rupture and crista reduction of some mitochondria,a large number of autophagosomes were observed,and the colocalization area percentage of TOMM20 and LC3 was increased.TOMM20 and cytochrome C oxidase subunit IV isoform 1(COX4I1)in hippocampus and selective autophagy adaptor protein 62(p62)protein expression,mitochondrial encoded ATP synthase 6(mt-ATP6)/Ribosomal protein L13(Rpl13)ratio and Na+-K+-ATPase content decreased,while PINK1 and Parkin protein expression,LC3-II/I ratio and ROS relative content increased,and the differences were statistically significant(t=4.602～52.012,all P<0.01).Compared with the model group,the neurological function score,cerebral infarction volume,pathological and ultrastructural damage of hippocampal neurons were significantly improved in the LIG low,high dose and NMDP groups,and the differences were statistically significant(t=4.851～12.525,all P<0.01).The colocalization of TOMM20 and LC3 and the content of Na+-K+-ATPase were increased,while the expression of TOMM20,COX4I1 and p62 proteins and the mt-ATP6/Rpl13 ratio were decreased in the high-dose LIG group.The protein expression of PINK1 and Parkin,LC3-II/I ratio and ROS relative content were increased,and the differences were statistically significant(t=4.087～33.211,all P<0.01).Compared with the LIG high-dose group,the Mdivi-1 and LIG+Mdivi-1 groups had significantly decreased colocalization of TOMM20 and LC3 and Na+-K+-ATPase content,and significantly increased expression of TOMM20,COX4I1 and p62 proteins and mt-ATP6/Rpl13 ratio.The protein expression of PINK1 and Parkin,LC3-II/I ratio and ROS relative content were decreased,and the differences were statistically significant(t=4.008～43.415,all P<0.01).However,the percentage of TOMM20 and LC3 co-localization area,PINK1 and Parkin protein expression,LC3-II/I ratio and Na+-K+-ATPase content in the hippocampus of the LIG+Mdivi-1 group were higher than those of the Mdivi-1 group.The protein expression of COX4I1 and p62,mt-ATP6/Rpl13 ratio and ROS level were lower than those in MDIV-1 group,and the differences were statistically significant(t=3.721～21.513,all P<0.01).Conclusion LIG may activate mitophagy by regulating PINK1/Parkin signaling pathway to protect neurons from cerebral ischemia-reperfusion injury in rats.

Objective:To evaluate the changes in cortical electroencephalogram (EEG) burst suppression rate (BSR) during sevoflurane anesthesia in septic mice and the correlation with cerebral blood perfusion.Methods:Forty SPF male C57BL/6J mice, aged 8-10 weeks, weighing 22-25 g, were divided into 2 groups ( n=20 each) by the random number table method: sham operation group (Sham group) and cecal ligation perforation group (CLP group). The sepsis model was established by cecal ligation and puncture in anesthetized animals. Mice in both groups inhaled 2% sevoflurane for 2 h. During sevoflurane anesthesia, BSR (30 min as an epoch) on electroencephalogram was recorded, and the cortical cerebral blood perfusion was recorded using the laser speckle flow imaging at 30, 60, 90 and 120 min of anesthesia. Results:Compared with Sham group, the cortical EEG BSR was significantly increased, and the cortical cerebral blood perfusion was decreased during sevoflurane anesthesia in CLP group ( P<0.05). Cortical EEG BSR was negatively correlated with cortical cerebral blood perfusion ( P<0.05). Conclusions:Cortical EEG BSR increases during sevoflurane anesthesia in septic mice, which may be related to decreased cortical cerebral blood perfusion.

Objective Nude mice xenograft model with aberrant activation of the PI3K/AKT pathway was established based on PIK3CA-overexpressing non-small cell lung cancer(NSCLC)cell lines PC-9,to observe the effect of Qingkailing injection combined with gefitinib on the growth of xenograft tumor in nude mice and explore its effects on ROS levels,mTOR pathway and STAT3 pathway.Methods After the xenografttumor model of BALB/c nude mice was established successfully,the mice were randomly divided into control group,Qingkailing injection group(10 mL·kg-1),gefitinib group(2.5 mg·kg-1),and Qingkailing combined with gefitinib group,with 5 mice in each group.They were administered for 32 days and then sacrificed.The tumor weight of each group was weighed and the tumor suppression rate was calculated;the level of ROS in the tumor tissues of each group was detected by flow cytometry;the protein levels of PI3K p110α,p-AKT and p-STAT3 in the xenograft tumor tissues of each group were detected by immunohistochemistry;the protein levels of the PI3K/AKT/mTOR pathway and the STAT3 pathway in the xenografttumor tissues of each group were detected by protein western blotting.Results Compared with the control group,Qingkailing injection could slow the tumor growth,significantly reduce the tumor weight,increase the level of ROS,and could significantly down-regulate the levels of PI3K p110α,AKT,mTOR,and STAT3 proteins in the tumor tissues(P<0.05);compared with the gefitinib single-agent group,the tumor growth of the Qingkailing combined with gefitinib group was slow,the weight of the tumors was significantly lower,and it also could significantly elevate the ROS level and downregulate the levels of PI3K p110α,AKT,mTOR,and STAT3 proteins in tumor tissues(P<0.05).Conclusion Qingkailing injection combined with gefitinib inhibited the growth of gefitinib-resistant xenograft in nude mice caused by abnormal activation of the PI3K/AKT pathway.Qingkailing injection may inhibit the PI3K/AKT pathway,its downstream mTOR pathway and STAT3 signaling pathway by up-regulating ROS levels,thereby enhancing the inhibitory effect of gefitinib on the proliferation of xenograft tumors of lung cancer xenografts in nude mice.

Freeze-drying technique,also known as lyophilization,is a process of removing moisture from a solution or suspension through freezing and vacuum dehydration to maintain the stability of the samples and prolong their shelf life.Freeze-drying technology has been widely used in food,pharmaceutical,clinical testing,chemical and other fields but its application in the field of forensic medicine has just started.Polymerase chain reaction(PCR)and its derivative detection technologies are widely used in forensic DNA detection,but PCR reagents need to be stored and transported at low temperature.In recent years,forensic scientists have begun applying freeze-drying technology to PCR amplification reagents to solve the transportation and storage problems of PCR reagents.In order to promote the application of PCR freeze-drying technology in forensic genetics,this paper mainly expounds the research course,system and process of PCR freeze-drying technology,compares the advantages and disadvantages of PCR reagents with traditional PCR reagents,and introduces the advantages and challenges of PCR freeze-drying reagents in forensic medicine.