Objective To characterize neurological alterations associated with structural bowel damage in patients with Crohn's disease(CD)through radiomics-assisted neurophenotyping,utilizing multiparametric brain MRI.Methods This prospective study enrolled patients with CD who underwent brain MRI,MR enterography,and ileocolonoscopy within one week.The Lémann Index was used to quantitatively assess cumulative structural bowel damage.CD patients were stratified into two groups based on a cutoff value of 4.8:those with bowel damage(LI>4.8)and those without bowel damage(LI≤4.8).A neurophenotype model was developed to characterize the neural changes associated with bowel damage in CD.Key features were selected from first-order features extracted from multiparametric brain MRI in the training cohort and validated in an independent test cohort.Results The final study population comprised 109 patients,including 51 individuals with bowel damage and 58 without bowel damage.The neurophenotype model scores were 0.785(95%CI:0.506～0.945)in the bowel damage group and 0.155(95%CI:0.093～0.394)in the non-bowel damage group,showing a statistically significant difference between the two groups(P<0.001).The developed model exhibited strong discriminative performance,with area under the receiver operating characteristic curve(AUC)values ranging from 0.824 to 0.918 across the training,vali-dation,and test cohorts(all P<0.05).Conclusion Our radiomics-assisted neurophenotype analysis reveals neural alterations in CD patients with bowel damage,which may indicate extraintestinal manifestations associated with cumulative intestinal injury.

Objective:This study aimed to explore the utility of transcutaneous electrical acupoint stimulation (TEAS) combined with skin sympathetic response (SSR) in assessing the effectiveness of perigastric autonomic nerve preservation during radical gastrectomy.Methods:A retrospective cohort analysis was conducted involving 221 patients who underwent laparoscopic radical gastrectomy at the Department of Gastric Surgery, Taizhou People's Hospital, affiliated with Nanjing Medical University, between June 2022 and September 2024. The cohort comprised 109 patients who underwent laparoscopic radical total gastrectomy without autonomic nerve preservation (total gastrectomy without nerve preservation group). Additionally, 112 patients underwent laparoscopic radical distal gastrectomy, including 34 patients who received autonomic nerve preservation (nerve preservation group) and 78 patients who did not (without nerve preservation group). TEAS was administered at the Zusanli and Tianshu acupoints one day before and one day after surgery, during which SSR latency and voltage amplitudes in the upper and lower extremities were recorded and compared across groups. Differences in SSR latency and voltage amplitude between the nerve preservation and non-nerve preservation groups of the distal gastrectomy cohort were also analyzed. Further, TEAS was applied at the same acupoints for 15 minutes on the 1st, 2nd, and 3rd postoperative days, and changes in intestinal sounds and intestinal functional recovery time were monitored. Surgical parameters, including operative duration, intraoperative blood loss, and harvested lymph node, were documented. Postoperative inflammatory indicators, including interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), and the incidence of anastomotic leakage, were evaluated. At three months postoperatively, gastroscopy was performed to assess residual gastric food and bile reflux. Additionally, the prognostic nutritional index (PNI) was evaluated across all patient groups.Results:Following total gastrectomy, TEAS of Zusanli combined with arms' SSR revealed a latency of (23 59.71±410.55) ms and a voltage amplitude of (0.43±1.67) mV; for the legs, latency was (2 596.88±369.01) ms and voltage amplitude was (0.25±0.08) mV. TEAS of Tianshu combined with arms' SSR demonstrated a latency of (2 746.47±224.37) ms and a voltage amplitude of (0.31±0.14) mV; for the legs, latency was (2 891.90±193.61) ms and voltage amplitude was (0.19±0.72) mV. Postoperative latency was significantly prolonged, and voltage amplitude was markedly reduced (all P < 0.01). In the distal gastrectomy with nerve preservation group, TEAS of Zusanli combined with arms' SSR showed a latency of (1 668.04±261.91) ms and a voltage amplitude of (0.78±0.26) mV; for the legs, latency was (1 568.86±220.09) ms and voltage amplitude was (0.61±0.24) mV. TEAS of Tianshu combined with arms' SSR demonstrated a latency of (1 519.36±206.99) ms and a voltage amplitude of (0.66±0.34) mV; for the legs, latency was (2 004.80±508.53) ms and voltage amplitude was (0.55±0.28) mV. In the distal gastrectomy without nerve preservation group, TEAS of Zusanli combined with arms' SSR revealed a latency of (2 385.95±710.27) ms and a voltage amplitude of (0.23±0.11) mV; for the legs, latency was (2 506.81±779.37) ms and voltage amplitude was (0.26±1.29) mV. TEAS of Tianshu combined with arms' SSR indicated a latency of (2 697.78±385.55) ms and a voltage amplitude of (0.21±0.14) mV; for the legs, latency was (2 949.14±506.61) ms and voltage amplitude was (0.17±0.11) mV. The group without nerve preservation exhibited significantly prolonged latencies and reduced voltage amplitudes (all P<0.01). No statistically significant differences were observed between the groups in operative time, intraoperative bleeding, the number of dissected lymph nodes, inflammatory indicators (IL-6, CRP, PCT) at 3 days postoperatively, or anastomotic leakage rates (all P>0.05). In the group without nerve preservation, bowel sounds on postoperative days 1, 2, and 3 were (0.36±0.58), (1.04±0.97), and (1.74±1.10) times/min, respectively, with bowel function recovery time of (62.24±9.91) hours. The PNI at 3 months postoperatively was (37.42±3.01). Incidences of food residue in the residual stomach and bile reflux were 21.79% (17/78) and 29.49% (23/78), respectively. In the group with nerve preservation, bowel sounds on postoperative days 1, 2, and 3 were (0.76±0.82), (2.03±1.34), and (3.71±1.27) times/min, respectively, with bowel function recovery time of (44.94±8.05) hours. The PNI at 3 months postoperatively was (41.34±3.40). Incidences of food residue and bile reflux were 5.88% (2/34) and 11.76% (4/34), respectively. Statistically significant differences were observed between the groups (all P < 0.05). Conclusion:TEAS of Zusanli and Tianshu combined with SSR provides an objective measure for assessing the preservation of perigastric autonomic nerves during radical gastrectomy.

Objective:To explore the clinical efficacy of double tract reconstruction and single flap valvuloplasty technique in laparoscopic proximal gastrectomy.Methods:A retrospective cohort study was adopted to analyze the clinical data of 65 patients with gastric cancer who underwent radical proximal gastrectomy at Taizhou People's Hospital Affiliated to Nanjing Medical University from July 2019 to April 2024. According to the different reconstruction methods, the patients were divided into the double tract reconstruction group (double tract; n=43) and oblique anastomosis of esophageal-gastric mucosal window with single flap valvuloplasty technique group (single flap n=22). The baseline data, surgical and postoperative recovery indicators, postoperative pathological results, gastroesophageal reflux at postoperative 6 months, and nutritional status at postoperative 1 year were compared between the two groups. Results:Comparisons of operative time, gastrointestinal reconstruction time, number of lymph nodes dissected, postoperative intestinal function recovery time, total protein, plasma albumin, hemoglobin, and lymphocyte count at 1 week postoperatively, prognostic nutritional index (PNI), time to normalization of postoperative white blood cell count and C-reactive protein, length of hospital stay, hospital costs, and incidence of postoperative pulmonary infection or anastomotic leakage between the two groups showed no statistically significant differences (all P>0.05). However, compared with the double tract group, the single muscle flap group had significantly higher intraoperative blood loss ( P<0.001), higher maximum postoperative body temperature ( P=0.004), and a significantly higher proportion of patients with pleural effusion ≥2 cm ( P=0.029).No statistically significant differences were observed between the two groups in terms of tumor length, length of esophageal involvement, Siewert classification, tumor differentiation degree, neural invasion, lymphovascular invasion, number of metastatic lymph nodes, tumor T stage and N stage, or UICC TNM staging for gastric cancer (all P>0.05). Nevertheless, the minimum distance of the lower resection margin in the double tract group was significantly longer than that in the single muscle flap group, with a statistically significant difference between the groups ( P<0.001). At 6 months postoperatively, results from the Quality of Life Questionnaire-Core 30 (QLQ-C30), Quality of Life Questionnaire-Stomach 22 (QLQ-ST022), Reflux Symptom Index scores, Visick grading, and gastroscopy (Los Angeles classification) all indicated that the incidence of reflux esophagitis in the double tract group was significantly lower than that in the single muscle flap group (all P<0.001). Gastrointestinal contrast examination showed no anastomotic stenosis in either group; gastroesophageal reflux occurred in 5 cases (11.6%) in the double tract group and 4 cases (18.2%) in the single muscle flap group, with no statistically significant difference (χ2=0.524, P=0.469). Gastroscopy results revealed that the incidence of reflux esophagitis at 6 months postoperatively was 9.3% (4/43) in the double tract group and 59.1% (13/22) in the single muscle flap group, with a statistically significant difference between the two groups (χ2=18.680, P<0.001).At 1 year postoperatively, the dual-chamber group showed better performance in body mass index(BMI), proportion of a decrease in BMI, plasma albumin, and PNI compared with the single muscle flap group, with statistically significant differences (all P<0.05). There were no statistically significant differences in hemoglobin or lymphocyte count between the two groups (all P>0.05). During 1 year of follow-up, one case of anastomotic recurrence occurred in each group, with no statistically significant difference between the groups ( P=0.624). Conclusions:Both proximal gastrectomy with double-tract anastomosis and esophagogastric mucosal window oblique anastomosis combined with single muscular flap valvuloplasty for digestive tract reconstruction are safe and feasible. However, double-tract anastomosis can better prevent the occurrence of postoperative reflux esophagitis, improve the patient's postoperative nutritional status, and enhance the patient's quality of life.

Objective To characterize neurological alterations associated with structural bowel damage in patients with Crohn's disease(CD)through radiomics-assisted neurophenotyping,utilizing multiparametric brain MRI.Methods This prospective study enrolled patients with CD who underwent brain MRI,MR enterography,and ileocolonoscopy within one week.The Lémann Index was used to quantitatively assess cumulative structural bowel damage.CD patients were stratified into two groups based on a cutoff value of 4.8:those with bowel damage(LI>4.8)and those without bowel damage(LI≤4.8).A neurophenotype model was developed to characterize the neural changes associated with bowel damage in CD.Key features were selected from first-order features extracted from multiparametric brain MRI in the training cohort and validated in an independent test cohort.Results The final study population comprised 109 patients,including 51 individuals with bowel damage and 58 without bowel damage.The neurophenotype model scores were 0.785(95%CI:0.506～0.945)in the bowel damage group and 0.155(95%CI:0.093～0.394)in the non-bowel damage group,showing a statistically significant difference between the two groups(P<0.001).The developed model exhibited strong discriminative performance,with area under the receiver operating characteristic curve(AUC)values ranging from 0.824 to 0.918 across the training,vali-dation,and test cohorts(all P<0.05).Conclusion Our radiomics-assisted neurophenotype analysis reveals neural alterations in CD patients with bowel damage,which may indicate extraintestinal manifestations associated with cumulative intestinal injury.

Objective:This study aimed to explore the utility of transcutaneous electrical acupoint stimulation (TEAS) combined with skin sympathetic response (SSR) in assessing the effectiveness of perigastric autonomic nerve preservation during radical gastrectomy.Methods:A retrospective cohort analysis was conducted involving 221 patients who underwent laparoscopic radical gastrectomy at the Department of Gastric Surgery, Taizhou People's Hospital, affiliated with Nanjing Medical University, between June 2022 and September 2024. The cohort comprised 109 patients who underwent laparoscopic radical total gastrectomy without autonomic nerve preservation (total gastrectomy without nerve preservation group). Additionally, 112 patients underwent laparoscopic radical distal gastrectomy, including 34 patients who received autonomic nerve preservation (nerve preservation group) and 78 patients who did not (without nerve preservation group). TEAS was administered at the Zusanli and Tianshu acupoints one day before and one day after surgery, during which SSR latency and voltage amplitudes in the upper and lower extremities were recorded and compared across groups. Differences in SSR latency and voltage amplitude between the nerve preservation and non-nerve preservation groups of the distal gastrectomy cohort were also analyzed. Further, TEAS was applied at the same acupoints for 15 minutes on the 1st, 2nd, and 3rd postoperative days, and changes in intestinal sounds and intestinal functional recovery time were monitored. Surgical parameters, including operative duration, intraoperative blood loss, and harvested lymph node, were documented. Postoperative inflammatory indicators, including interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), and the incidence of anastomotic leakage, were evaluated. At three months postoperatively, gastroscopy was performed to assess residual gastric food and bile reflux. Additionally, the prognostic nutritional index (PNI) was evaluated across all patient groups.Results:Following total gastrectomy, TEAS of Zusanli combined with arms' SSR revealed a latency of (23 59.71±410.55) ms and a voltage amplitude of (0.43±1.67) mV; for the legs, latency was (2 596.88±369.01) ms and voltage amplitude was (0.25±0.08) mV. TEAS of Tianshu combined with arms' SSR demonstrated a latency of (2 746.47±224.37) ms and a voltage amplitude of (0.31±0.14) mV; for the legs, latency was (2 891.90±193.61) ms and voltage amplitude was (0.19±0.72) mV. Postoperative latency was significantly prolonged, and voltage amplitude was markedly reduced (all P < 0.01). In the distal gastrectomy with nerve preservation group, TEAS of Zusanli combined with arms' SSR showed a latency of (1 668.04±261.91) ms and a voltage amplitude of (0.78±0.26) mV; for the legs, latency was (1 568.86±220.09) ms and voltage amplitude was (0.61±0.24) mV. TEAS of Tianshu combined with arms' SSR demonstrated a latency of (1 519.36±206.99) ms and a voltage amplitude of (0.66±0.34) mV; for the legs, latency was (2 004.80±508.53) ms and voltage amplitude was (0.55±0.28) mV. In the distal gastrectomy without nerve preservation group, TEAS of Zusanli combined with arms' SSR revealed a latency of (2 385.95±710.27) ms and a voltage amplitude of (0.23±0.11) mV; for the legs, latency was (2 506.81±779.37) ms and voltage amplitude was (0.26±1.29) mV. TEAS of Tianshu combined with arms' SSR indicated a latency of (2 697.78±385.55) ms and a voltage amplitude of (0.21±0.14) mV; for the legs, latency was (2 949.14±506.61) ms and voltage amplitude was (0.17±0.11) mV. The group without nerve preservation exhibited significantly prolonged latencies and reduced voltage amplitudes (all P<0.01). No statistically significant differences were observed between the groups in operative time, intraoperative bleeding, the number of dissected lymph nodes, inflammatory indicators (IL-6, CRP, PCT) at 3 days postoperatively, or anastomotic leakage rates (all P>0.05). In the group without nerve preservation, bowel sounds on postoperative days 1, 2, and 3 were (0.36±0.58), (1.04±0.97), and (1.74±1.10) times/min, respectively, with bowel function recovery time of (62.24±9.91) hours. The PNI at 3 months postoperatively was (37.42±3.01). Incidences of food residue in the residual stomach and bile reflux were 21.79% (17/78) and 29.49% (23/78), respectively. In the group with nerve preservation, bowel sounds on postoperative days 1, 2, and 3 were (0.76±0.82), (2.03±1.34), and (3.71±1.27) times/min, respectively, with bowel function recovery time of (44.94±8.05) hours. The PNI at 3 months postoperatively was (41.34±3.40). Incidences of food residue and bile reflux were 5.88% (2/34) and 11.76% (4/34), respectively. Statistically significant differences were observed between the groups (all P < 0.05). Conclusion:TEAS of Zusanli and Tianshu combined with SSR provides an objective measure for assessing the preservation of perigastric autonomic nerves during radical gastrectomy.

Objective:To explore the clinical efficacy of double tract reconstruction and single flap valvuloplasty technique in laparoscopic proximal gastrectomy.Methods:A retrospective cohort study was adopted to analyze the clinical data of 65 patients with gastric cancer who underwent radical proximal gastrectomy at Taizhou People's Hospital Affiliated to Nanjing Medical University from July 2019 to April 2024. According to the different reconstruction methods, the patients were divided into the double tract reconstruction group (double tract; n=43) and oblique anastomosis of esophageal-gastric mucosal window with single flap valvuloplasty technique group (single flap n=22). The baseline data, surgical and postoperative recovery indicators, postoperative pathological results, gastroesophageal reflux at postoperative 6 months, and nutritional status at postoperative 1 year were compared between the two groups. Results:Comparisons of operative time, gastrointestinal reconstruction time, number of lymph nodes dissected, postoperative intestinal function recovery time, total protein, plasma albumin, hemoglobin, and lymphocyte count at 1 week postoperatively, prognostic nutritional index (PNI), time to normalization of postoperative white blood cell count and C-reactive protein, length of hospital stay, hospital costs, and incidence of postoperative pulmonary infection or anastomotic leakage between the two groups showed no statistically significant differences (all P>0.05). However, compared with the double tract group, the single muscle flap group had significantly higher intraoperative blood loss ( P<0.001), higher maximum postoperative body temperature ( P=0.004), and a significantly higher proportion of patients with pleural effusion ≥2 cm ( P=0.029).No statistically significant differences were observed between the two groups in terms of tumor length, length of esophageal involvement, Siewert classification, tumor differentiation degree, neural invasion, lymphovascular invasion, number of metastatic lymph nodes, tumor T stage and N stage, or UICC TNM staging for gastric cancer (all P>0.05). Nevertheless, the minimum distance of the lower resection margin in the double tract group was significantly longer than that in the single muscle flap group, with a statistically significant difference between the groups ( P<0.001). At 6 months postoperatively, results from the Quality of Life Questionnaire-Core 30 (QLQ-C30), Quality of Life Questionnaire-Stomach 22 (QLQ-ST022), Reflux Symptom Index scores, Visick grading, and gastroscopy (Los Angeles classification) all indicated that the incidence of reflux esophagitis in the double tract group was significantly lower than that in the single muscle flap group (all P<0.001). Gastrointestinal contrast examination showed no anastomotic stenosis in either group; gastroesophageal reflux occurred in 5 cases (11.6%) in the double tract group and 4 cases (18.2%) in the single muscle flap group, with no statistically significant difference (χ2=0.524, P=0.469). Gastroscopy results revealed that the incidence of reflux esophagitis at 6 months postoperatively was 9.3% (4/43) in the double tract group and 59.1% (13/22) in the single muscle flap group, with a statistically significant difference between the two groups (χ2=18.680, P<0.001).At 1 year postoperatively, the dual-chamber group showed better performance in body mass index(BMI), proportion of a decrease in BMI, plasma albumin, and PNI compared with the single muscle flap group, with statistically significant differences (all P<0.05). There were no statistically significant differences in hemoglobin or lymphocyte count between the two groups (all P>0.05). During 1 year of follow-up, one case of anastomotic recurrence occurred in each group, with no statistically significant difference between the groups ( P=0.624). Conclusions:Both proximal gastrectomy with double-tract anastomosis and esophagogastric mucosal window oblique anastomosis combined with single muscular flap valvuloplasty for digestive tract reconstruction are safe and feasible. However, double-tract anastomosis can better prevent the occurrence of postoperative reflux esophagitis, improve the patient's postoperative nutritional status, and enhance the patient's quality of life.

AIM:To investigate the molecular mechanism underlying ferroptosis induced by celastrol(Cel)in huamn pancreatic cancer cell line PANC-1.METHODS:The viability of PANC-1 cells was analyzed by MTT assay,and the effects of Cel on cell proliferation were analyzed using EdU and colony formation assays.Flow cytometry and fluores-cence microscopy were used to assess and observe changes in lipid reactive oxygen species(ROS)levels,respectively,while the levels of malondialdehyde(MDA),glutathione(GSH)and Fe2+were measured using specific kits.The protein expression of glutathione peroxidase 4(GPX4)was evaluated by Western blot,and GPX4 ubiquitination was measured by immunoprecipitation.RESULTS:It was found that the viability,proliferation and colony formation in PANC-1 cells de-creased gradually as the concentration of Cel increased.Addition of Cel alone to the cells reduced both cell rounding and viability,while treatment with ferrostatin-1(Fer-1)alone or in combination with Cel had no effect on either cell morpholo-gy or viability.Fluorescence staining of lipid ROS with BODIPYTM 581/591 C11 followed by flow cytometry analysis showed significantly increased levels of green fluorescence indicative of oxidized lipid ROS,which were further increased after treatment of the cells with Cel.Treatment of the cells with both Cel and Fer-1 reduced the green fluorescence and lip-id ROS levels.Treatment with Cel also increased the levels of MDA and Fe2+,relative to the controls,which reducing the levels of GSH,while addition of both Cel and Fer-1 to the cells restored the levels of MDA,Fe2+,and GSH to those of the control group.CONCLUSION:Treatment with Cel reduces the proliferation of pancreatic cancer cells by inducing fer-roptosis through promoting the ubiquitination and degradation of GPX4.

AIM:This study aimed to explore the induction of ferroptosis in non-small-cell lung cancer(NSCLC)cells by tubeimoside II(TBMS II)and to elucidate the underlying molecular mechanisms.METHODS:H460 NSCLC cells were cultured in vitro.Cell survival rates were assessed by using MTT assays,and doses of TBMS II resulting in below 50%survival were selected for further experimentation.Cell migration was evaluated using Transwell assays and the effects of TBMS II on H460 cell proliferation were assessed by colony formation assays.Flow cytometry and fluores-cence microscopy were used to assess changes in lipid peroxidation(lipid ROS),and the levels of GSH,T-AOC,MDA,and Fe2+were measured using commercial kits.Protein levels of GPX4,SLC7A11,FTH1,NCOA4,P62,and LC3 were examined using Western blot.Changes in mitochondrial structure were detected by transmission electron microscopy,and immunofluorescence was used to assess LC3 co-localization of FTH1 and NCOA4,as well as co-localization of LC3 and NCOA4 with lysosomes.RESULTS:Compared with the control group,TBMS II dose-dependently reduced H460 cell via-bility,migration,and clone formation,accompanied by the appearance of vacuoles within the cells.TBMS II treatment al-so led to decreased GSH and T-AOC levels,while increasing the cellular contents of MDA,indicating oxidative stress.Ad-ditionally,there was a decrease in the expression of the antioxidant proteins SLC7A11 and GPX4 in the cells,while lipid ROS and Fe2+levels were increased in proportion to the TBMS II concentration.The ferroptosis inhibitor ferrostatin-1 re-versed cell death caused by TBMS II,suggesting ferroptosis induction.Furthermore,increasing the TBMS II concentra-tion resulted in an upregulation of the autophagy marker proteins LC3 II/LC3 I and P62,indicative of increased autopha-gy.TBMS II also affected mitochondrial morphology in the cells,as seen in reduced mitochondrial fluorescence intensity.Protein expression of NCOA4 increased with higher TBMS II concentrations,while that of FTH1 decreased.Co-localiza-tion of LC3 II with FTH1 and NCOA4,as well as the lysosomal association of LC3 II and FTH1,also increased in a dose-dependent manner.CONCLUSION:TBMS II induces ferritinophagy in H460 cells,leading to decreased cell viability and increased ferroptosis.

Objective To investigate the effect of MACC1 on RSL3-induced ferroptosis in colorectal cancer cells and explore its molecular mechanism.Methods MACC1 expression was detected in SW620,HCT116,LOVO and RKO cells using Western blotting.The effects of different concentrations of RSL3(an inducer of ferroptosis)or Fer-1(an inhibitor of ferroptosis)alone,or 10 μmol/L RLS3 combined with 10 μmol/L Fer-1,on viability of SW620 cells were examined using MTT assay.The survival of SW620 cells with mRNA interference of MACC1 was analyzed following treatment with RSL3,and RT-qPCR and Western blotting were performed to detect the changes in MACC1 expressions after RSL3 treatment at different concentrations and the changes in GPX4 expression after MACC1 knockdown.Flow cytometry and laser confocal microscopy were used to analyze the changes in ROS-induced lipid peroxidation in SW620 cells after MACC1 knockdown.Results SW620 cells had the highest MACC1 expression among the 4 colorectal cancer cell lines.Treatment with RSL3 significantly inhibited the viability of SW620 cells in a dose-dependent manner,while Fer-1 did not significantly affect the survival of SW620 cells.RSL3 alone reduced SW620 cell survival by 50%(P<0.01),and the combined treatment with RSL3 and Fer-1 caused no significant changes in cell survival(P>0.05).Treatment with RSL3 concentration-dependently suppressed MACC1 expressions at both the mRNA and protein levels in SW620 cells(P<0.01).MACC1 knockdown obviously enhanced the cytotoxic effect of RSL3,inhibited the expression of GPX4,and increased ROS-induced lipid peroxidation in SW620 cells(P<0.05).Conclusion MACC1 knockdown enhances RSL3-induced ferroptosis in cultured colorectal cancer cells by inhibiting the expression of GPX4.

Objective To investigate the effect of MACC1 on RSL3-induced ferroptosis in colorectal cancer cells and explore its molecular mechanism.Methods MACC1 expression was detected in SW620,HCT116,LOVO and RKO cells using Western blotting.The effects of different concentrations of RSL3(an inducer of ferroptosis)or Fer-1(an inhibitor of ferroptosis)alone,or 10 μmol/L RLS3 combined with 10 μmol/L Fer-1,on viability of SW620 cells were examined using MTT assay.The survival of SW620 cells with mRNA interference of MACC1 was analyzed following treatment with RSL3,and RT-qPCR and Western blotting were performed to detect the changes in MACC1 expressions after RSL3 treatment at different concentrations and the changes in GPX4 expression after MACC1 knockdown.Flow cytometry and laser confocal microscopy were used to analyze the changes in ROS-induced lipid peroxidation in SW620 cells after MACC1 knockdown.Results SW620 cells had the highest MACC1 expression among the 4 colorectal cancer cell lines.Treatment with RSL3 significantly inhibited the viability of SW620 cells in a dose-dependent manner,while Fer-1 did not significantly affect the survival of SW620 cells.RSL3 alone reduced SW620 cell survival by 50%(P<0.01),and the combined treatment with RSL3 and Fer-1 caused no significant changes in cell survival(P>0.05).Treatment with RSL3 concentration-dependently suppressed MACC1 expressions at both the mRNA and protein levels in SW620 cells(P<0.01).MACC1 knockdown obviously enhanced the cytotoxic effect of RSL3,inhibited the expression of GPX4,and increased ROS-induced lipid peroxidation in SW620 cells(P<0.05).Conclusion MACC1 knockdown enhances RSL3-induced ferroptosis in cultured colorectal cancer cells by inhibiting the expression of GPX4.