Objective To investigate the role of quercetin(QUE)in ferroptosis during intestinal ischemia-reperfusion(IR)injury and elucidate its underlying mechanisms.Methods ① Potential target genes of QUE were predicted using the TCMSP,PharmMapper,and SwissTargetPredictive databases.Target genes associated with intestinal IR injury and ferroptosis were collected from GeneCards,PharmGKB,and OMIM databases.After overlapping genes were identified and analyzed,protein-protein interaction(PPI)networks were constructed using the STRING database and then visualized with Cytoscape 3.10.0.Molecular docking was performed to validate the binding conformations between QUE and key targets.② In vivo experiments were conducted to verify QUE's protective effects against intestinal IR injury.Thirty-six SPF-grade male C57BL/6J mice(6～8 weeks old,body weight:22±2 g)were randomly divided into Sham,Sham+QUE,IR,IR+QUE,IR+QUE+erastin(IR+QUE+Era),and IR+QUE+kevetrin hydrochloride(IR+QUE+KH)groups,with 6 mice in each group.Mouse model of intestinal IR injury was induced by 45 min ischemia of the superior mesenteric artery followed by 60 min reperfusion.HE staining was used to observe histopathological changes in the intestinal tissues.ELISA was employed to the serum or intestinal contents of diamine oxidase(DAO),pro-inflammatory cytokines(TNF-α,IL-6,IL-1β),and ferroptosis markers[glutathione(GSH)and Fe2+].Western blotting was utilized to detect the protein expression of glutathione peroxidase 4(GPX4),acyl-CoA synthetase long-chain family member 4(ACSL4),and tumor protein 53(p53).Results ① Network pharmacology identified 460 QUE targets,1 552 intestinal IR injury targets,and 1 967 ferroptosis-related targets,and 92 overlapping genes were identified as potential therapeutic targets.Molecular docking revealed a strong binding affinity between QUE and p53(binding energy:-6.8 kcal/mol).② In vivo experiments demonstrated that the IR+QUE group exhibited reduced intestinal damage and lower Chiu's score(P<0.05),decreased serum DAO content but elevated intestinal DAO content(P<0.05),decreased levels of TNF-α,IL-6,and IL-1β in the serum and intestinal tissues(P<0.05),reduced Fe2+accumulation,and increased GSH content(P<0.05),and up-regulated GPX4(P<0.05)and down-regulated ACSL4 and p53 expression(P<0.05)at protein level when compared with the IR group.While,the administration of ferroptosis agonist Era,or p53 agonist KH resulted in diminished therapeutic effects of QUE(P<0.05)when compared with the IR+QUE group.Conclusion QUE alleviates intestinal IR injury by inhibiting ferroptosis,which may be associated with its down-regulating p53 expression.

Objective To investigate whether remimazolam attenuates intestinal ischemia-reperfusion(I/R)injury in mice by regulating ferroptosis through connexin-43(CX43).Methods Molecular docking was applied to predict the binding affinity of remimazolam to CX43.A total of 72 SPF-grade adult male C57BL/6J mice(6～8 weeks old,weighing 20～25 g)were subjected.Thirty of them were randomly divided into sham operation group(Sham group),I/R group 1,and I/R+10,20 and 40 mg/kg remimazolam groups(RM10,RM20 and RM40 groups),with 6 mice in each group.Another 30 mice were randomly assigned into 5 groups(n=6),I/R group 2,erastin group(E group),I/R+40 mg/kg remimazolam group 2(RM40 group 2),I/R+Fer-1 group(Fer-1 group),and erastin+40 mg/kg remimazolam group(ERM group).The left 12 mice were randomly and equally grouped into I/R+RM+oe-NC group(oe-NC group)and I/R+RM+oe-CX43 group(oe-CX43 group).The Fer-1 group was given an intraperitoneal injection of 5 mg/kg Fer-1 in 1 h prior to reperfusion,the E group was given 10 mg/kg erastin intraperitoneally 1 d before modeling,and all the remimazolam groups,the oe-NC group and the oe-CX43 group were injected intravenously with corresponding doses of remimazolam 30 min pre-modeling,while the oe-NC and oe-CX43 groups were injected with empty vector virus and overexpression of CX43 vector virus,respectively,48 h before the administration of remimazolam.A mouse intestinal I/R injury model was constructed by clamping the superior mesenteric artery for 45 min and reperfusion for 30 min.The small intestine tissues were harvested and observed for pathological changes,and the intestinal mucosal damage was assessed with Chiu's score.The contents of Fe2+,total iron,malondialdehyde(MDA),glutathione(GSH),and superoxide dismutase(SOD)were detected by colorimetric assay;the production of reactive oxygen species(ROS)was determined by DHE probe;the expression of ferroptosis-related genes was determined by RT-qPCR;and the expression levels of CX43,GPX4,and SLC7A11 were detected by Western blotting.Results Molecular docking indicated that remimazolam had a binding energy of-6.699 kcal/mol with CX43 protein,suggesting good binding affinity between them.Compared with the Sham group,the I/R group 1 showed increases in Chiu's scores and CX43 expression(P<0.05),along with pathological damage to intestinal tissues,and elevated contents of Fe2+,total iron,ROS and MDA(P<0.05),and down-regulated GPX4 and SLC7A11(P<0.05).Compared with the I/R group 1,Chiu's score was reduced in the RM40 group,and CX43 was significantly down-regulated(P<0.05),contents of Fe2+,total iron,ROS,and MDA were decreased(P<0.05),and expression levels of GPX4 and SLC7A11 were enhanced(P<0.05),and severity of intestinal histological damage was attenuated in both the RM40 and Fer-1 groups.Compared with the E group,the ERM group had the decreases in CX43 expression level(P<0.05),Fe2+,total iron,ROS,and MDA contents(P<0.05),and increases in GPX4 and SLC7A11 expression levels(P<0.05),with the improvement in intestinal tissue.Compared with the oe-NC group,overexpression of CX43 resulted in the increased CX43 expression,elevated contents of Fe2+,total iron,ROS and MDA(P<0.05)and decreased expression of GPX4 and SLC7A11(P<0.05),leading to the exacerbated injury in intestinal tissue.Conclusion Remimazolam attenuates intestinal I/R injury by inhibiting ferroptosis through down-regulating CX43 expression.

Objective To investigate the anesthetic effects and adverse effects of cypropofol and propofol combined with alfentanil,respectively,for gastroenteroscopy.Methods A total of 162 patients who underwent elective gastroenteroscopy at the Gastrointestinal Endoscopy Center of the First Hospital of Lanzhou University from January to February 2024 were enrolled,including 86 males and 76 females,at an age of 18～65 years old,with a BMI value of 18～30 kg/m2,and ASA grade ≤ Ⅱ.They were randomly divided into propofol group(Group P)and cypropofol group(Group C),with 81 cases in each group.All patients were sedated with 0.7 μg/kg alfentanil,and in 30 s later,2 mg/kg propofol and 0.4 mg/kg cypropofol was intravenously dripped into Group P and Group C,respectively.When the modified alertness/sedation score(MOAA/S)≤1,a gastroscope was started to insert.The related indicators,including total procedure time,successful cases of sedation,induction time and awakening time,heart rate,blood pressure,and pulse oximetry saturation were recorded,occurrence of adverse reactions such as hypotension,respiratory depression,injection pain,intraoperative body movement,nausea and vomiting were observed,and the satisfaction of endoscopists and of patients to anesthesia were recorded and compared between the 2 groups.Results There were no statistical differences in the success rate of sedation,induction time and awakening time between the 2 groups.The patients of the Group C had more stable intraoperative vital signs,statistically lower incidences of injection pain,respiratory depression and hypotension(P＜0.05),and increased satisfaction for anesthesia(P＜0.05)when compared with those in Group P.No obvious difference were observed in the satisfaction of endoscopist to anesthesia between the 2 groups.Conclusion In combination with small-dose alfentanil,0.4 mg/kg cypropofol shows similar sedation effect as 2 mg/kg propofol in gastroenteroscopy,with comparable induction and awakening time.Cypropofol has more advantages in stable intraoperative vital signs,less adverse effects such as low blood pressure,respiratory depression and injection pain,higher the patient satisfaction,which is worthy of clinical promotion.

Objective To systematically evaluate the efficacy and safety of ciprofol for sedation and anesthesia outside the operating room.Methods Databases such as PubMed,Embase,Cochrane Library,Web of Science,CNKI,Wanfang Data,CBM,and VIP were searched for randomized controlled trials(RCTs)related to the efficacy and safety of ciprofol for sedation and anesthesia outside the operating room.The search covered all publications up to June 2023.Statistical analysis was performed using RevMan 5.4 software and Stata 15.0.Results Twelve RCTs were included,involving 2 192 patients,of which 1 154 were in the ciprofol group and 1 038 in the propofol group.Compared with the propofol group,the anesthesia induction time(MD=0.28 min,95％CI 0.08-0.47 min,P=0.006)and recovery time(MD=1.16 min,95％CI 0.44-1.87 min,P=0.001)were significantly longer in the ciprofol group,and the inci-dences of injection pain(OR=0.04,95％CI 0.02-0.06,P＜0.001),hypotension(OR=0.64,95％CI 0.49-0.83,P=0.0008),hypoxemia(OR=0.44,95％CI 0.21-0.91,P=0.03),and respirato-ry depression(OR=0.19,95％CI 0.11-0.32,P＜0.001)were significantly lower.There were no sta-tistically significant differences between the two groups in terms of sedation success rate,physician satisfac-tion,the difference in heart rate before and after anesthesia induction,incidence of body movement,brady-cardia,nausea and vomiting,and dizziness.Conclusion The anesthetic effect of cyclopofol and propofol is similar when used for anesthesia outside the operating room.Compared to propofol,ciprofol offers comparable anesthetic effects for sedation and anesthesia outside the operating room,with a lesser impact on respiratory function and more stable hemodynamics.Ciprofol also significantly lowers the incidence of adverse reactions such as injection pain,hypotension,hypoxemia,and respiratory depression.

ObjectiveTo investigate the mechanism of astragaloside-Ⅳ （AS-Ⅳ） in regulating pyroptosis to alleviate intestinal ischemia-reperfusion injury （IRI） by combining network pharmacology and in vivo experiments. MethodFirstly， the corresponding target genes of AS-Ⅳ were obtained from TraditionalChineseMedicineSystemsPharmacology（TCMSP） database and Swiss Target Prediction database， and the target genes related to intestinal IRI and Pyroptosis were obtained from GeneCards database， and the common target genes of the three were obtained by drawing Venn diagrams through unspiralized website. Protein-protein interaction （PPI） network was constructed by STRING database and Cytoscape software to screen common target genes and imported into Cytoscape software to obtain core target genes. Microbiotics platform was used for gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） enrichment analysis and prediction of the mechanism of action of AS-Ⅳ in regulating Pyroptosis to alleviate intestinal IRI. Then C57/BL6J mice were randomly divided into 5 groups normal group， model group（IR）， drug administration group （IR+AS-Ⅳ）， nucleotide-binding oligomerization structural domain-like receptor protein 3 （NLRP3） agonist NSS group （IR+AS-Ⅳ+NSS）， and NLRP3 inhibitor MCC950 group （IR+AS-Ⅳ+MCC950） by using a randomized numerical table method. The intestinal IRI model was established by clamping the superior mesenteric artery for 45 min and resuming perfusion for 2 h in the model group， the drug administration group， the NLRP3 agonist NSS group， and the NLRP3 inhibitor MCC950 group， and the normal group was only separated from the vessels without clamping. The administration group， the NLRP3 agonist NSS group， and the NLRP3 inhibitor MCC950 group were gavaged with astragaloside dissolved in 0.1% dimethylsulfoxide （50 mg·kg^-1） for 3 consecutive days before modeling， with the last gavage 2 h before modeling， and the remaining two groups were gavaged with equal amounts of saline. The NLRP3 agonist NSS group was injected intraperitoneally with 4 mg·kg^-1 of NSS 1 h before modeling， and the NLRP3 inhibitor MCC950 group was injected intraperitoneally with 10 mg·kg^-1 of MCC950 1 h before modeling.The mice were put to death by reperfusion for 2 h， and intestinal tissues were obtained. The levels of IL-18 and IL-1β were detected by enzyme linked immunosorbent assay（ELISA）， and the protein expression of thioredoxin-binding protein （TXNIP）， NLRP3， Caspase-1 and pyrocatechin D （GSDMD） were detected by Western blot， and the pathological changes of intestinal tissues were evaluated by Chiu's score. ResultNetwork pharmacological analysis showed that there were 1599 targets of intestinal IRI， 199 targets of AS-Ⅳ action， 197 targets of pyroptosis， and 20 targets common to all three. There were 10 core targets， including NLRP3， TXNIP， silencing information regulator 1 （SIRT1）， high mobility group protein 1 （HMGB1）， interleukin-18 （IL-18）， GSDMD， and metallo matrix protease-9 （MMP-9），et al. The results of in vivo experiments showed that compared with the normal group， Chiu's score was elevated in the model group， the levels of IL-18，IL-1β inflammatory factors in mouse intestinal tissues were elevated （P<0.05）， and the protein expression levels of TXNIP， NLRP3， Caspase-1， and GSDMD were elevated （P<0.05）. Compared with the model group，Chiu's score was decreased in the administered group and NLRP3 inhibitor MCC950 group，the level of IL-18，IL-1β inflammatory factors in the intestinal tissue of mice was decreased（P<0.05）， and the level of TXNIP，NLRP3，Caspase-1，GSDMD protein expression was decreased（P<0.05）. Compared with the administered group， Chiu's score was elevated in the NLRP3 agonist NSS group， the levels of IL-18， IL-1β inflammatory factors in mouse intestinal tissues were elevated （P<0.05）， and the protein expression levels of NLRP3， Caspase-1， and GSDMD were elevated （P<0.05）. Compared with the NLRP3 inhibitor MCC950 group， the NLRP3 agonist NSS group had elevated Chiu's scores， elevated levels of IL-18，IL-1β inflammatory factors in mouse intestinal tissues （P<0.05）， and elevated levels of TXNIP，NLRP3， Caspase-1， and GSDMD protein expression （P<0.05）. ConclusionNetwork pharmacological predictions were consistent with the results of in vivo experiments， and astragaloside attenuated intestinal ischemia-reperfusion injury by inhibiting cellular pyroptosis through the TXNIP-NLRP3 signaling pathway.

Objective:To investigate the effect of dexmedetomidine(DEX)on the polarization of alveolar macrophages in-duced by lipopolysaccharide(LPS)and to explore the related mechanisms.Methods:Rat alveolar macrophages NR8383 were cul-tured in vitro.Experiment one was divided into control group,model group(1 μg/ml LPS),DEX low,medium and high dose groups(1,5,10 mg/kg DEX+10 mg/kg LPS).Experiment two was divided into DEX high dose group(10 mg/kg)and DEX high dose+Colive-lin(JAK2/STAT3 signaling pathway activator)group(10 mg/kg DEX+0.5 μmol/L Colivelin).The morphological changes of rat alveo-lar macrophages NR8383 were observed by inverted microscope;RT-PCR method was used to detect the expression levels of iNOS and Arg1 mRNA in NR8383 cells,and flow cytometry was used to detect the expression levels of CD86 and CD163 proteins in NR8383 cells;Western blot was used to detect the expression levels of surface marker proteins TNF-α,iNOS,SOCS,Arg1,TGF-β and JAK2/STAT3 signaling pathway related proteins in NR8383 cells.Results:Compared with control group,there were a lot of cell debris in the intercellular space of NR8383 in the model group,the proportions of iNOS mRNA,CD86 positive cells,and the expression levels of TNF-α,p-JAK2/JAK2,p-STAT3/STAT3 were significantly increased,the proportions of Arg1 mRNA,CD163 positive cells,and the expression levels of SOCS and TGF-β were significantly reduced(P<0.05);compared with the model group,the NR8383 intercellular cell debris in the DEX low,medium,and high dose groups were decreased,the proportions of iNOS mRNA,CD86 positive cells,and the expression levels of TNF-α,p-JAK2/JAK2,p-STAT3/STAT3 were significantly reduced,the proportions of Arg1 mRNA,CD163 positive cells,and the expression levels of SOCS and TGF-β were significantly increased(P<0.05).The reactivation of the JAK2/STAT3 signal pathway by Colivelin could weaken the role of DEX in LPS induced NR8383 cell polarization.Conclusion:DEX can inhibit the M1 polarization of NR8383 cells induced by LPS,which may be achieved by inhibiting the JAK2/STAT3 signaling pathway.

AIM: To explore schisandrin B (Sch B) pretreatment reduces intestinal ischemia reperfusion injury (IIRI) through inhibiting apoptosis by activation of Nrf2/HO-1 signing pathway in mice by network pharmacology and in vivo experiment. METHODS: (1) The targets of Sch B and IIRI were searched from online databases, Drawing Venn diagram to obtain the common target of them. Cytoscape software was imported to construct the protein-protein interaction (PPI) network to establish the "Drugs-Disease-core target gene" network. The mechanism of Sch B against IIRI was predicted through GO and KEGG enrichment analysis. (2) Thirty-six C57BL/6J mice were randomly divided into six groups (n = 6). The model of IIRI was established in four groups except the sham operation group. Three of the groups were pretreated with Sch B, Nrf2 inhibitor ML385, and Sch B + ML385, respectively. After the experiment, intestinal tissue samples were taken for HE staining, Chiu ' s score, apoptosis staining, immunohistochemistry (IHC), and immunoblotting (Western blot). RESULTS: A total of 412 Sch B related tar- gets, 2 166 IIRI related targets and 153 common targets were screened out through network pharmacology. There were 88 "Sch B-IIRI-core target gene" included NFE2L2 (Nrf2), HMOX1 (HO-1), BCL2, CASP3 (caspase 3), and so on. KEGG enrichment analysis screened 163 related pathways, apoptosis pathway ranked high showing that the pathway may play a key role in the treatment of IIRI by Sch B. The animal experiment had shown that Sch B reduced the Chiu's score and apoptotic while upregulating Nrf2, HO-1, Bcl-2 protein expression levels and Bcl-2/Bax, downregulating Bax, and cleaved caspase-3 expression levels, thereby reducing IIRI in mice, and that Nrf2 inhibitor ML385 reversed this process (P < 0.05). CONCLUSION: This study reveals that Sch B has the characteristics of multi-target and multi-pathway in the reduction of IIRI, and Sch B can reduce IIRI through inhibiting apoptosis by activation of Nrf2/ HO-1 pathway.

AIM: To verify the role of tetramethylpyrazine (TMP) in intestinal ischemia-reperfusion (I/ R) injury and its relationship with pyroptosis. METHODS: Thirty-six healthy SPF male C57BL / 6 mice, 8-12 weeks old, weighing 20-25 g, were divided into six groups randomized by table of random number (n = 6/group): Sham group (S1 group)Ischemia/reperfusion group (I/R1 group), I/R + TMP treatment group: 15 mg/kg (T15 group), 30 mg/kg (T30 group), 60 mg/kg (T60-1 group), 120 mg/kg (T120 group). In experiment 2, thirty healthy SPF male C57BL/6 mice were divided into five groups (n = 6/group): Sham group (S2 group), I/R group (I/ R2 group), I/R + dimethyl sulphoxide (DMSO) group (DMSO group), I/R + TMP (60 mg/kg) group (T60-2 group), and I/R + DMSO + TMP (60 mg/kg) + Nigericin sodium salt (NSS) group (T60+NSS group). I/R-induced intestinal injury was established by clamping the superior mesenteric artery for 45 minutes, followed by 120 minutes of reperfusion, while the sham group mice underwent isolation of superior mesenteric artery without clamping. An NLRP3 agonist NSS was dissolved in DMSO, was intraperitoneally injected (4 mg/kg) 60 minutes before ischemia. And DMSO group mice were intraperitoneally administered with corresponding DMSO. Different TMP dosage groups and T60+NSS group mice were intraperitoneally administered with TMP 30 minutes before ischemia. IL-1β and IL-18 concentrations in the intestine were measured at 120 minutes after reperfusion by ELISA. The pathological changes of the sections were observed by optical microscope, and the intestinal mucosal injury was evaluated by Chiu's score grading. Western blot was used to detect NLRP3, Caspase-1, and GSDMD in intestinal tissue. RESULTS: Statistically significant increase of Chiu's score, IL-1β, IL-18 concentrations in the I/R1 group were found as compared with S1 group (P<0.05). And compared with I / R1 group, Chiu's score and IL-1β, IL-18 concentrations in the T60-1, T120 groups were reduced (P<0.05). Moreover, Chiu's score in the T120 group was lower than that in the T60 group (P<0.05). We found a statistically significant increase of Chiu's score and IL-1β, IL-18 concentrations and the expression of NLRP3, GSDMD, caspase-1 in the I/R group (P<0.05) as compared with S2 group. Compared with I / R2 group, Chiu's score, IL-1β, IL-18 concentrations and NLRP3, GSDMD, caspase-1 expression in the T60-2 group was reduced (P<0.05). Compared with T60-2 group, Chiu's score, IL-1β, IL-18 concentrations and NLRP3, GSDMD, caspase-1 expression in the T60 + NSS group were upregulated (P<0.05). CONCLUSION: The protective effect of TMP against intestinal I / R injury was dose-dependent. And TMP can decrease pyroptosis mainly by inhibiting the activation of the NLRP3 inflammasome.

Objective:To evaluate the effect of dexmedetomidine on the thioredoxin-interacting protein (TXNIP)/apoptosis signal-regulated kinase 1 (ASK1) signaling pathway in a mouse model of intestinal ischemia-reperfusion (I/R).Methods:Thirty-two SPF healthy adult male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 4 groups ( n=8 each) using a random number table method: sham operation group (Sham group), intestinal I/R group (I/R group), TXNIP inhibitor resveratrol group (Res group) and dexmedetomidine group (Dex group). The mouse model of intestinal I/R injury was developed by clamping the superior mesenteric artery for 45 min followed by 120-min reperfusion in anesthetized animals. Resveratrol 30 mg/kg was intraperitoneally injected before developing the model in Res group, and dexmedetomidine 25 μg/kg was intraperitoneally injected at 30 min before ischemia in Dex group. Blood samples were collected by cardiac puncture at the end of 120-min reperfusion, then the mice were sacrificed, and the small intestine tissues were removed for microscopic examination and for determination of the serum diamine oxidase (DAO) concentration (by enzyme-linked immunosorbent assay) and expression of TXNIP, ASK1 and cleaved-caspase-3 in small intestinal tissues (by Western blot). The apoptosis rate of intestinal epithelial cells was calculated. The intestinal damage was assessed and scored according to Chiu. Results:Compared with group Sham, the Chiu′s score, serum DAO concentrations and apoptosis rate of intestinal epithelial cells were significantly increased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was up-regulated in group I/R ( P<0.05). Compared with group I/R, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in group Res ( P<0.05). Compared with I/R group, the Chiu′s score, serum DAO concentration and apoptosis rate of intestinal epithelial cells were significantly decreased, and the expression of TXNIP, ASK-1 and cleaved-caspase-3 was down-regulated in Dex group ( P<0.05). Conclusions:The mechanism by which dexmedetomidine alleviates intestinal I/R injury may be related to inhibition of the TXNIP/ASK1 signaling pathway and reduction of cell apoptosis in mice.

Objective:To evaluate the safety and efficacy of ciprofol and propofol for gynecological surgeries with general anesthesia through a meta-analysis.Methods:Electronic databases including PubMed, Embase, Cochrane, Web of Science, China National Knowledge Infrastructure, Wanfang Data, China Biomedical Literature Database, and China Science and Technology Journal Database were searched for randomized controlled trials comparing the safety and efficacy of ciprofol and propofol in gynecological surgeries with general anesthesia from inception to May 2023. Meta-analysis was performed using Revman 5.4 software.Results:Six randomized controlled trials were included, involving 741 patients, of which 371 received ciprofol and 370 received propofol. Compared with propofol group, the emergence time was significantly prolonged, the difference in mean arterial blood pressure, systolic blood pressure and diastolic blood pressure before and after anesthesia induction was decreased, and the incidence of injection pain, respiratory depression, body movement and hypotension was decreased in ciprofol group ( P<0.05). There were no significant differences between the two groups in terms of time of successful anesthesia induction, difference in BIS values and heart rate before and after anesthesia induction, and incidence of tachycardia, bradycardia and hypertension ( P>0.05). Conclusions:Ciprofol is comparable to propofol in terms of efficacy and has better safety than propofol when used in gynecologic surgeries with general anesthesia.