AIM:This study aims to investigate whether vitamin E succinate(VES)induces autophagy in hu-man gastric cancer cells through the promotion of mitochondria-associated endoplasmic reticulum membranes(MAMs).METHODS:Human gastric cancer cell lines MKN28 and MKN45 were cultured in vitro.Cell viability was assessed us-ing the CCK8 assay,and two cell growth curves were plotted to determine the treatment concentration of VES.Control groups,VES dose groups(MKN28:5,10,20,and 40 mg/L;MKN45:10,20,40,and 80 mg/L),an autophagy-posi-tive control group(rapamycin,RAPA,100 nmol/L),and a MAMs-positive control group(oligomycin A,10 mg/L)were set up.Cells were harvested after 24 h of treatment for subsequent experiments.The formation of autophagosomes and MAMs was observed using transmission electron microscopy.The expression levels of autophagy-related proteins,includ-ing beclin-1,LC3-II/LC3-I,and p62,were detected by Western blot.MAMs labeled with split green fluorescent protein(GFP)were visualized by fluorescence microscopy.The expression of mitofusin 2(MFN2),a key molecule of MAMs,was also detected by Western blot.To inhibit MFN2 specifically,the cells were treated with mitochondrial fusion inhibitor 8(MFI8)and simultaneously transfected with an MFN2 plasmid to achieve MFN2 overexpression(OE-MFN2).The cells were divided into control group,MFI8(20 μmol/L)group,VES groups(20 mg/L for MKN28 cells and 40 mg/L for MKN45 cells),VES+MFI8 group,OE-MFN2+MFI8 group and OE-MFN2+VES+MFI8 group.The MAMs were visualized by fluorescence microscopy,and the expression changes of MFN2,beclin-1 and LC3-II/I were detected by Western blot.RESULTS:The results of the CCK8 assay showed that VES significantly inhibited the viability of both human gastric can-cer cell lines(P<0.05).After VES treatment,the formation of typical autophagosomes and MAMs was observed in both cell lines by transmission electron microscopy.Fluorescence microscopy showed a significant increase in GFP signals of MAMs.Western blot analysis showed that with increasing doses of VES,the expression levels of MFN2,beclin-1,and LC3-II/I were significantly up-regulated,while that of p62 was significantly down-regulated(P<0.05).Compared with VES group,the cells pretreated with MFI8 followed by VES exposure showed markedly reduced GFP signals of MAMs and much lower protein levels of MFN2,beclin-1 and LC3-II/LC3-I(P<0.05).Transfection with an MFN2 overexpression plasmid rescued MFN2 expression.Compared with VES+MFI8 group,the cells in OE-MFN2+VES+MFI8 group had much higher protein expression levels of MFN2,beclin-1 and LC3-II/LC3-I(P<0.05).CONCLUSION:The VES may partici-pates in the regulation of autophagy in human gastric cancer cells by promoting the formation of MAMs.

AIM:This study aims to investigate whether vitamin E succinate(VES)induces autophagy in hu-man gastric cancer cells through the promotion of mitochondria-associated endoplasmic reticulum membranes(MAMs).METHODS:Human gastric cancer cell lines MKN28 and MKN45 were cultured in vitro.Cell viability was assessed us-ing the CCK8 assay,and two cell growth curves were plotted to determine the treatment concentration of VES.Control groups,VES dose groups(MKN28:5,10,20,and 40 mg/L;MKN45:10,20,40,and 80 mg/L),an autophagy-posi-tive control group(rapamycin,RAPA,100 nmol/L),and a MAMs-positive control group(oligomycin A,10 mg/L)were set up.Cells were harvested after 24 h of treatment for subsequent experiments.The formation of autophagosomes and MAMs was observed using transmission electron microscopy.The expression levels of autophagy-related proteins,includ-ing beclin-1,LC3-II/LC3-I,and p62,were detected by Western blot.MAMs labeled with split green fluorescent protein(GFP)were visualized by fluorescence microscopy.The expression of mitofusin 2(MFN2),a key molecule of MAMs,was also detected by Western blot.To inhibit MFN2 specifically,the cells were treated with mitochondrial fusion inhibitor 8(MFI8)and simultaneously transfected with an MFN2 plasmid to achieve MFN2 overexpression(OE-MFN2).The cells were divided into control group,MFI8(20 μmol/L)group,VES groups(20 mg/L for MKN28 cells and 40 mg/L for MKN45 cells),VES+MFI8 group,OE-MFN2+MFI8 group and OE-MFN2+VES+MFI8 group.The MAMs were visualized by fluorescence microscopy,and the expression changes of MFN2,beclin-1 and LC3-II/I were detected by Western blot.RESULTS:The results of the CCK8 assay showed that VES significantly inhibited the viability of both human gastric can-cer cell lines(P<0.05).After VES treatment,the formation of typical autophagosomes and MAMs was observed in both cell lines by transmission electron microscopy.Fluorescence microscopy showed a significant increase in GFP signals of MAMs.Western blot analysis showed that with increasing doses of VES,the expression levels of MFN2,beclin-1,and LC3-II/I were significantly up-regulated,while that of p62 was significantly down-regulated(P<0.05).Compared with VES group,the cells pretreated with MFI8 followed by VES exposure showed markedly reduced GFP signals of MAMs and much lower protein levels of MFN2,beclin-1 and LC3-II/LC3-I(P<0.05).Transfection with an MFN2 overexpression plasmid rescued MFN2 expression.Compared with VES+MFI8 group,the cells in OE-MFN2+VES+MFI8 group had much higher protein expression levels of MFN2,beclin-1 and LC3-II/LC3-I(P<0.05).CONCLUSION:The VES may partici-pates in the regulation of autophagy in human gastric cancer cells by promoting the formation of MAMs.

Objective:To investigate the effect of dexmedetomidine (DEX) on reducing urethral stimulation in patients undergoing laparoscopic gastrointestinal surgery.Methods:From January 2019 to February 2020, 90 patients undergoing elective laparoscopic gastrointestinal surgery under general anesthesia in the Third Affiliated Hospital of Sun Yat-sen University were selected. They were randomly divided into 3 groups: catheterization before induction (group A), catheterization during induction (group B), and catheterization after induction (group C). In group A, patients received general anesthesia after awake catheterization. In group B, intravenous injection of DEX 0.5 μg/kg was pumped for 10 minutes, followed by catheterization and induction. In group C, patients received general anesthesia and then catheterization. Visual analogue scale (VAS) score of urethral stimulation, morphine dosage and the incidence of agitation during resuscitation were recorded. The heart rate and mean arterial pressure of the three groups were compared at the time of entering the room, catheterization, tracheal intubation, entering postanesthesia care unit (PACU), about extubation and 30 minutes after extubation.Results:The fluctuation of blood pressure and heart rate in group B was significantly less than that in group A and group C at the time of extubation and 30 minutes after extubation ( P<0.05). VAS of urethral stimulation in group B [(2.9±0.9)point] was significantly lower than that in group A [(4.4±1.8)point] when catheter was indwelling ( P<0.05). After extubation, VAS in group B [(2.8±1.1)point] was significantly lower than that in group A [(3.2±1.2)point] and C [(5.2±1.8)point] ( P<0.05). The utilization rate of morphine within 24 hours after surgery in group B (10%) was significantly lower than that in the other two groups (40%, 57%), and the incidence of postoperative agitation in group A and B was lower than that in group C within PACU ( P<0.05). The satisfaction of patients in group B (86.7%) was higher than that in group A (70%) and C (46.7%). The satisfaction of PACU personnel in group A (76.7%) and B (80%) was significantly higher than that in group C (43.3%). Conclusions:Sedation with dexmedetomidine during urethral catheterization can reduce urethral stimulation during resuscitation and improve patients' and PACU staffs' satisfaction.

BACKGROUND:The effect of advanced glycation end products (AGEs) on bone resorption is controversial. Our previous study has shown that bone resorption is significantly inhibited when AGEs present with pre-osteoclast cells RAW 264.7, while the effect of AGEs on osteoclastic acidification remains unknown. OBJECTIVE:To investigate the effect of AGEs on osteoclastic acidification and the underlying mechanism. METHODS:RAW 264.7 cells were induced by RANKL (15μg/L;normal group) to generate osteoclasts, and AGEs (50-400 mg/L;experimental group) or bovine serum albumin (100 mg/L;control group) were added at the beginning of the induction. The effect of AGEs on bone resorption was evaIuated by anaIyzing the area of bone resorption on the Osteo Assay Surface plates, and the effect of AGEs on osteoclastic acidification was evaluated by acridine orange staining. Furthermore, the expression levels of V-ATPase a3 and CIC-7 were detected to investigate the underlying mechanism. RESULTS AND CONCLUSION:The bone resorption area in the AGEs group was significantly decreased compared with the normal group (P<0.05). Acridine orange staining reveaIed that the red fluorescence (620 nm) intensity in the AGEs group was significantly decreased compared with the normal group (P<0.05), and this inhibitory effect became obvious with the increase of AGEs concentration. Immunocytochemistry, western blot assay and PCR findings showed that the expression levels of V-ATPase a3 and CIC-7 in the AGEs group were decreased significantly compared with the normal group (P<0.05). To conclude, AGEs exert inhibitory effect on osteoclastic acidification, probably by inhibiting the expression of V-ATPase a3 and CIC-7.

Objective To investigate personal identification of mixed seminal stain of two individuals, we combined the detection of genotyping autosomal, Y and X STR and sequencing mtDNA hypervariable Ⅰ (HV Ⅰ ) region. Methods We analyzed autosomal, Y and X STR with commercial kit and separating and sequencing HVⅠfragments of mixed seminal stain from two males by SSCP electrophoresis. Results Four genetic markers of the high amount sample can be obtained when mixed ratio is more than 1:10. When the proportion of two samples is close, the suspect could be excluded or, to some extent, identified by comparing with our results. Conclusion The combined detection of four genetic marker systems can, to some degree, solve the personal identification from mixed seminal stain of two individuals.

BACKGROUND:The effects of advanced glycation end products (AGEs) on osteoclast-induced bone resorption is controversial and the underlying mechanisms remain unclear. Most of the studies indicate that AGEs can enhance bone resorption, while some othersshowthe opposite effects.
OBJECTIVE:To investigate the effects of AGEs on osteoclast-induced inorganicmatrixdissolution and organic componentdegradation and the underlying mechanisms.
METHODS:RAW 264.7 cels were induced to generate osteoclasts,and AGEs (50-400 μg/mL) or control-bovine serum albumin (100 μg/mL) was added since the beginning of the induction. The effect of AGEs on bone resorption was evaluated by analyzing the area of resorption pits on the Osteo Assay Surface plates and the expression of cathepsin K. Furthermore, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cels, nuclei per osteoclasts and the expression of integrinανβ3were detected.
RESULTS AND CONCLUSION:The area of resorption pits and expression of cathepsin K in AGEs groups were significantly decreased compared withthecontrol group, and this inhibiting effect became more obvious with the increase of AGEs concentration. TRAP staining also showed that number of TRAP-positivemultinucleated celsand nuclei per osteoclast were significantly reduced in an AGE dose-dependent manner. Quantitative PCR revealed that the expression of integrin ανβ3decreased significantly with the extension of AGEs incubation time. These data indicate that AGEs can exert inhibitory effects on organic and inorganicmatrixdegradation induced by osteoclasts. The underlying mechanism may be involved in the inhibitory effects of AGEs on directed differentiation and cel fusion of osteoclast precursor cels, and migration and adhension of osteoclasts.