Objective To investigate the effects of harmine(HM)on the expression level of mitochondrion fusion related proteins and mitochondrial function injury in PC 12 cells.Methods PC 12 cells were divided into cell control group,HM group,mitochondrion mitosis inhibitor Mdivi-1 group,HM+Mdivi-1 group,mitochondrion fission agonist WY14643 group,HM+WY14643 group,with drug concentrations of 1,10,25,50,100 μmol·L-1.After 24 h treatment,the MTT method was used to detect the cell survival rate,and a microscope was used to observe the cell morphology,MitoTracker Red probe staining was used to observe the mitochondrial morphology and the length ratio of vertical and horizontal axes,JC-1 staining was used to detect the mitochondrial membrane potential,and a kit was used to detect ATP level and lactate dehydrogenase(LDH)activity.Immunofluorescence staining and Western blotting were used to assess the expression levels of caspase-3,apoptosis-promoting protein(Bax)cytochrome C(cyt-c),mitochondrial fusion protein(Mfn2)and mitochondrial mitotic protein(Drp-1).The interference sequence of Drp1 was transfected by electroporation,and the siRNA sequence with good transfection effect was screened.The related indicators were detected by fluorescence method,MTT method,and immunoblotting method in cooperation with drug intervention.Results MTT results showed that compared with the cell control group,the survival rate of HM group,Mdivi-1 group,HM+Mdivi-1 group,WY14643 group and HM+WY14643 group decreased significantly(P＜0.01),and the EC50 were(11.48±2.32),(12.35±1.67),(14.88±2.07),(39.14±3.25),(20.09±1.97),respectively.According to this,subsequent experiments selected 20 μmol·L-1for HM,WY 14643 and HM+WY14643 as working concentrations to construct PC 12 cell model.Microscopic observation and MitoTracker Red probe staining showed that the cell density in the drug group decreased in varying degrees,and a transition from branched to round morphology in the drug-treated groups was observed.The morphology of mitochondria tended to be round,and the ratio of the length of the longitudinal axis to transverse axis was(3.33±0.72)in the cell control group,(2.19±0.58)in the HM group,(2.45±0.44)in Mdivi-1 group,and(1.43±0.62)in HM+Mdivi-1 group,respectively.The results of JC-1 staining showed that compared with the cell control group,the mitochondrial mode potential of the HM group significantly decreased(P＜0.01).ROS significantly increased(P＜0.01)and ATP levels decreased(P＜0.01),and LDH enzyme activity increased(P＜0.01).Immunofluorescence staining and Western blotting results showed that compared with the cell control group,the expression levels of proapoptotic proteins Bax,cytochrome C,and caspase-3 in the HM group were significantly increased(all P＜0.01).Compared with the cell control group,the expression level of mitochondrial fission related protein Drp1 in HM group was significantly higher(P＜0.01).The expression level of mitochondrial fusion related protein Mfn2 significantly decreased(P＜0.01).After specific interference with Drp1 and synergistic intervention with HM,the survival rate of PC 12 cells in each interference group decreased compared to each drug intervention group.The expression of Drp1 and Mfn2 was downregulated,and the differences were statistically significant(P＜0.05 or P＜0.01).Conclusion HM can reduce the mitochoudrial membrane potential and ATP levels by accumulating ROS,there by activating the caspase-3 apoptosis pathway and promoting cell apoptosis.Mitochondrial fusion division may be involved in the damage of PC12 cells caused by HM,initiating apoptosis through the mitochondrial pathway.

Objective:To investigate the effect of immune checkpoint inhibitors on reducing residual lymph node metastasis in patients with gastric cancer.Methods:The cohort of this retrospective study comprised patients from Nanfang Hospital of Southern Medical University and the First Affiliated Hospital of Xiamen University who had undergone systemic treatment prior to gastrectomy with D2 lymphadenectomy and had achieved Grade 1 primary tumor regression (TRG1) from January 2014 to December 2023. After exclusion of patients who had undergone preoperative radiotherapy, data of 58 patients (Nanfang Hospital: 46; First Affiliated Hospital of Xiamen University: 12) were analyzed. These patients were allocated to preoperative chemotherapy (Chemotherapy group, N=36 cases) and preoperative immunotherapy plus chemotherapy groups (Immunotherapy group, N=22 cases). There were no significant differences between these groups in sex, age, body mass index, diabetes, tumor location, pathological type, Lauren classification, tumor differentiation, pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, mismatch repair protein status, number of preoperative treatment cycles, or duration of preoperative treatment (all P>0.05). The primary outcome measure was postoperative lymph node downstaging. Secondary outcomes included postoperative depth of invasion by tumor, number of lymph nodes examined, and factors affecting residual lymph node metastasis status. Results:Lymph node downstaging was achieved significantly more often in the Immunotherapy group than the Chemotherapy group (pN0: 90.9% [20/22] vs. 61.1% [22/36]; pN1: 4.5% [1/22] vs. 36.1% [13/36]; pN2: 4.5% [1/22) vs. 0; pN3: 0 vs. 2.8% [1/36], Z=-2.315, P=0.021). There were no significant difference between the two groups in number of lymph nodes examined (40.5±16.3 vs. 40.8±17.5, t=0.076, P=0.940) or postoperative depth of invasion by primary tumor (pT1a: 50.0% [11/22] vs. 30.6% [11/36]; pT1b: 13.6% [3/22] vs. 19.4% [7/36]; pT2: 13.6% [3/22] vs. 13.9% [5/36]; pT3: 13.6% [3/22] vs. 25.0% [9/36]; pT4a: 9.1% [2/22] vs. 11.1% [4/36], Z=-1.331, P=0.183). Univariate analysis revealed that both preoperative treatment regimens were associated with residual lymph node metastasis status in patients whose primary tumor regression was TRG1 (χ 2=6.070, P=0.014). Multivariate analysis incorporated the following factors: pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, number of preoperative treatment cycles, and preoperative treatment duration. We found that a combination of immunotherapy and chemotherapy administered preoperatively was an independent protective factor for reducing residual lymph node metastases in study patients whose primary tumor regression was TRG1 (OR=0.147, 95%CI: 0.026–0.828, P=0.030). Conclusion:Compared with preoperative chemotherapy alone, a combination of preoperative immunotherapy and chemotherapy achieved greater reduction of residual lymph node metastases in the study patients who achieved TRG1 tumor regression in their primary lesions.

Objective:To investigate the effect of immune checkpoint inhibitors on reducing residual lymph node metastasis in patients with gastric cancer.Methods:The cohort of this retrospective study comprised patients from Nanfang Hospital of Southern Medical University and the First Affiliated Hospital of Xiamen University who had undergone systemic treatment prior to gastrectomy with D2 lymphadenectomy and had achieved Grade 1 primary tumor regression (TRG1) from January 2014 to December 2023. After exclusion of patients who had undergone preoperative radiotherapy, data of 58 patients (Nanfang Hospital: 46; First Affiliated Hospital of Xiamen University: 12) were analyzed. These patients were allocated to preoperative chemotherapy (Chemotherapy group, N=36 cases) and preoperative immunotherapy plus chemotherapy groups (Immunotherapy group, N=22 cases). There were no significant differences between these groups in sex, age, body mass index, diabetes, tumor location, pathological type, Lauren classification, tumor differentiation, pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, mismatch repair protein status, number of preoperative treatment cycles, or duration of preoperative treatment (all P>0.05). The primary outcome measure was postoperative lymph node downstaging. Secondary outcomes included postoperative depth of invasion by tumor, number of lymph nodes examined, and factors affecting residual lymph node metastasis status. Results:Lymph node downstaging was achieved significantly more often in the Immunotherapy group than the Chemotherapy group (pN0: 90.9% [20/22] vs. 61.1% [22/36]; pN1: 4.5% [1/22] vs. 36.1% [13/36]; pN2: 4.5% [1/22) vs. 0; pN3: 0 vs. 2.8% [1/36], Z=-2.315, P=0.021). There were no significant difference between the two groups in number of lymph nodes examined (40.5±16.3 vs. 40.8±17.5, t=0.076, P=0.940) or postoperative depth of invasion by primary tumor (pT1a: 50.0% [11/22] vs. 30.6% [11/36]; pT1b: 13.6% [3/22] vs. 19.4% [7/36]; pT2: 13.6% [3/22] vs. 13.9% [5/36]; pT3: 13.6% [3/22] vs. 25.0% [9/36]; pT4a: 9.1% [2/22] vs. 11.1% [4/36], Z=-1.331, P=0.183). Univariate analysis revealed that both preoperative treatment regimens were associated with residual lymph node metastasis status in patients whose primary tumor regression was TRG1 (χ 2=6.070, P=0.014). Multivariate analysis incorporated the following factors: pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, number of preoperative treatment cycles, and preoperative treatment duration. We found that a combination of immunotherapy and chemotherapy administered preoperatively was an independent protective factor for reducing residual lymph node metastases in study patients whose primary tumor regression was TRG1 (OR=0.147, 95%CI: 0.026–0.828, P=0.030). Conclusion:Compared with preoperative chemotherapy alone, a combination of preoperative immunotherapy and chemotherapy achieved greater reduction of residual lymph node metastases in the study patients who achieved TRG1 tumor regression in their primary lesions.

Background::Previous studies have revealed that diabetes mellitus (DM) promotes disease progress of gastric cancer (GC). This study aimed to further investigating whether DM advanced lymph nodes (LNs) metastasis in GC.Methods::The clinicopathologic data of GC patients with >15 examined LN (ELN) between October 2004 and December 2019 from a prospectively maintained database were included. The observational outcomes included the number (N3b status) and anatomical distribution (N3 stations) of metastatic LN (MLN).Results::A total of 2142 eligible patients were included in the study between October 2004 and December 2019. N3 stations metastasis (26.8% in DM vs. 19.3% in non-DM, P = 0.026) and N3b status (18.8% in DM vs. 12.8% in non-DM, P = 0.039) were more advanced in the DM group, and multivariate logistic regression analyses confirmed that DM was an independent factor of developing N3 stations metastasis (odds ratio [OR] = 1.771, P= 0.011) and N3b status (OR= 1.752, P= 0.028). Also, multivariate analyses determined DM was independently associated with more MLN (β = 1.424, P = 0.047). The preponderance of N3 stations metastasis (DM vs. non-DM, T1-2: 2.2% vs. 4.9%, T3: 29.0% vs. 20.3%, T4a: 38.9% vs. 25.8%, T4b: 50.0% vs. 36.6%; ELN16-29: 8.6% vs. 10.4%, ELN30-44: 27.9% vs. 20.5%, ELN ≥ 45: 37.7% vs. 25.3%), N3b status (DM vs. non-DM, T1-2: 0% vs. 1.7%, T3: 16.1% vs. 5.1%, T4a: 27.8% vs. 19.1%, T4b: 44.0% vs. 28.0%; ELN16-29: 8.6% vs. 7.9%, ELN30-44: 18.0% vs. 11.8%, ELN ≥ 45: 26.4% vs. 17.3%), and the number of MLN (DM vs. non-DM, T1-2: 0.4 vs. 1.1, T3: 8.6 vs. 5.2, T4a: 9.7 vs. 8.6, T4b: 17.0 vs. 12.8; ELN16-29: 3.6 vs. 4.6, ELN30-44: 5.8 vs. 5.5, ELN ≥ 45: 12.0 vs. 7.7) of DM group increased with the advancement of primary tumor depth stage and raising of ELN. Conclusions::DM was an independent risk factor for promoting LN metastasis. The preponderance of LN involvement in the DM group was aggravated with the advancement of tumor depth.

The prognosis of advanced gastric cancer (AGC) is extremely poor. There is no standard and satisfactory treatment strategy for AGC. In clinical practice, some AGC patients can achieve long-term survival. However, it is not clear which type of AGC can benefit the best in specific treatment mode. Because of the high heterogeneity of AGC, it is particularly important to further dig out more significant beneficiary groups. Therefore, experts put forward the classification based on the biological characteristics and the surgery-oriented classification to predict and select patients who benefit from conversion therapy. While for immunotherapy, the biomarkers, molecular subtyping and potential combination strategies are explored to break through its bottleneck in the treatment in AGC that only certain individuals benefit from it. The authors review the research progress in treatment for advanced gastric cancer.

Objective:To explore the characteristics and advantages of Whole-Mounting immnunofluorescent staining and laser speckle flow imaging technology in the vascular imaging of mouse ear extended flap.Methods:In this study, total of 25 ICR mice were included.Ten ICR mice were cut off the middle and lateral angiosome to establish an extended flap model, and 3 days later, the changes in the blood supply of the ear flap were observed. The ear area , tissue layer thickness and blood vessel distribution in the healthy side were observed at the same time.Obtain the mouse ears 3 days after modeling, and dissect them into three layers, i.e, the anterior skin layer , the cartilage layer and the posterior skin layer. The distribution and morphology of blood vessels, nerves and monocytes/macrophages in the anterior skin layer were stained and detected by the whole-mount immunofluorescence staining.Ten mice were adopted and an incision was made through the ear horizontally above the bifurcation of the middle angiosome of the mouse ear to establish a delayed extended flap model. Then the blood flow changes in the mouse ear were observed by laser speckle flow imaging andt he blood perfusion values were recorded immediately, 1 d, 2 d, 3 d and 4 d after the operation, respectively.Results:The area of the mouse ear was about 1.3 cm 2 , the thickness was about (0.16±0.04) mm, and the blood was supplied by three vascular bundles: the lateral caudal vascular bundle, the middle vascular bundle and the medial cephalic vascular bundle. The thickness of the anterior and posterior skin and cartilage of the mouse ears were (88±5)μm, (41±3)μm and (29±2)μm, respectively. The whole-mount immunofluorescence staining results clearly showed that the diameter of small vessels in the choke area was (50 ± 6) μm on the third day after modeling. It could be seen that the nerve and artery in mouse ear were in concomitant relationship and the nerve segment attached to the surface of the artery without obvious accompany or clinging to the vein. There were a large number of monocyte macrophages distributed in clusters in the dilated and curved arteries, but they were only scattered outside the artery. Laser speckle flow imaging results showed that there were (6 ± 2) transverse vessels in each auricular flap, and the diameter and blood flow increased significantly in the delayed extended earflap model. Immediately after the operation and at 1d, 2d, 3d, and 4d, the average blood perfusion values of transverse vessels were (92±11) PU, (136±26) PU, (147±27) PU and (176±27) PU, respectively. Conclusions:The Whole-Mounting immnunofluorescent staining and the laser speckle blood flow imaging technology can be used to well observe the blood vessels, nerves, mononuclear macrophages and blood flow perfusion of the mouse extended flap, which can play an important role in the study of blood supply of mouse extended flap.

Objective:To explore the characteristics and advantages of Whole-Mounting immnunofluorescent staining and laser speckle flow imaging technology in the vascular imaging of mouse ear extended flap.Methods:In this study, total of 25 ICR mice were included.Ten ICR mice were cut off the middle and lateral angiosome to establish an extended flap model, and 3 days later, the changes in the blood supply of the ear flap were observed. The ear area , tissue layer thickness and blood vessel distribution in the healthy side were observed at the same time.Obtain the mouse ears 3 days after modeling, and dissect them into three layers, i.e, the anterior skin layer , the cartilage layer and the posterior skin layer. The distribution and morphology of blood vessels, nerves and monocytes/macrophages in the anterior skin layer were stained and detected by the whole-mount immunofluorescence staining.Ten mice were adopted and an incision was made through the ear horizontally above the bifurcation of the middle angiosome of the mouse ear to establish a delayed extended flap model. Then the blood flow changes in the mouse ear were observed by laser speckle flow imaging andt he blood perfusion values were recorded immediately, 1 d, 2 d, 3 d and 4 d after the operation, respectively.Results:The area of the mouse ear was about 1.3 cm 2 , the thickness was about (0.16±0.04) mm, and the blood was supplied by three vascular bundles: the lateral caudal vascular bundle, the middle vascular bundle and the medial cephalic vascular bundle. The thickness of the anterior and posterior skin and cartilage of the mouse ears were (88±5)μm, (41±3)μm and (29±2)μm, respectively. The whole-mount immunofluorescence staining results clearly showed that the diameter of small vessels in the choke area was (50 ± 6) μm on the third day after modeling. It could be seen that the nerve and artery in mouse ear were in concomitant relationship and the nerve segment attached to the surface of the artery without obvious accompany or clinging to the vein. There were a large number of monocyte macrophages distributed in clusters in the dilated and curved arteries, but they were only scattered outside the artery. Laser speckle flow imaging results showed that there were (6 ± 2) transverse vessels in each auricular flap, and the diameter and blood flow increased significantly in the delayed extended earflap model. Immediately after the operation and at 1d, 2d, 3d, and 4d, the average blood perfusion values of transverse vessels were (92±11) PU, (136±26) PU, (147±27) PU and (176±27) PU, respectively. Conclusions:The Whole-Mounting immnunofluorescent staining and the laser speckle blood flow imaging technology can be used to well observe the blood vessels, nerves, mononuclear macrophages and blood flow perfusion of the mouse extended flap, which can play an important role in the study of blood supply of mouse extended flap.

Advanced gastric cancer (AGC) has a high recurrence rate (especially peritoneal relapse) and a poor prognosis. Systematic chemotherapy or targeted therapy have not been able to significantly reduce the major cause of an unfavorable prognosis, namely the high peritoneal AGC recurrence rate post-surgery. Further studies concerning the application of hyperthermic intraperitoneal chemotherapy (HIPEC) post curative surgery for AGC patients, namely the prophylactic HIPEC (P-HIPEC), have involved a prophylactic approach to prevent peritoneal relapse following curative gastrectomy in high-risk patients. Theoretically, breaking the "plasma-peritoneal barrier" increases cytotoxic chemotherapy activity via a synergistic hyperthermic effect; therefore, HIPEC can eradicate free cancer cells and micro-metastasis within the peritoneal cavity intraoperatively or soon after curative gastrectomy to reduce peritoneal recurrence. Many clinical trials have shown that P-HIPEC can reduce peritoneal recurrence and improve prognosis of AGC patients. However, some studies applying HIPEC at an early stage have revealed a high rate of complications that limited generalizability. This procedure has been increasingly adopted, given the complication rate has now been reduced and safety has been proven. Recently, for assessing the important role of HIPEC, many high-quality prospective randomized controlled clinical trials have been conducted to further investigate the best guidance for P-HIPEC and to demonstrate its effectiveness and safety with a higher grade of evidence. With theory development, the technique, equipment, and management of HIPEC and the role of P-HIPEC for AGC continues to evolve. This study summarizes the progress of P-HIPEC for high-risk AGC patients.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Chemotherapy, Cancer, Regional Perfusion ; Combined Modality Therapy ; Humans ; Hyperthermia, Induced ; Neoplasm Recurrence, Local ; Peritoneal Neoplasms ; drug therapy ; Randomized Controlled Trials as Topic ; Stomach Neoplasms ; drug therapy

Conversion therapy is adopted to achieve radical cure for patients with originally unresectable but potentially resectable late stage gastric cancer, who obtain partial or complete remission after systemic chemotherapy, to acquire relatively longer postoperative survival and recurrence-free survival. Some of the previous researches on conversion therapy for originally unresectable gastric cancer suggest that high chemotherapy response rate, high pathological response rate and R0 resection rate are associated with favorable prognosis. And the efficacy of patients with lymphatic metastasis is better than that of those with peritoneal metastasis. The protocol of conversional chemotherapy varies and so does its efficacy according to different reports. Latest clinical researches indicate that initially unresectable gastric cancer gained higher remission rate and better chance of R0 operation and consequently prolonged survival from paclitaxel based triplet chemotherapy. However, not all originally unresectable gastric cancer can benefit from conversion therapy due to the high heterogeneity of its biological behavior. Regarding the enormous number of originally unresectable gastric cancer patients, it will be a research hot spot in the field of surgical oncology, on screening criteria to select cases suitable for conversion. Exploration on conversion therapy for gastric cancer is still at initial stage, and reports that have been published are mostly single-centered with limited sample, lacking of sufficient evidence on its feasibility, safety and efficacy. Expert consensus on conversion indication, case selection, chemotherapy regimen, efficacy assessment and resection range is absent. So it is in urgent need for higher level clinical evidence to support and guide this practice. Such goal can never be achieved without joint efforts of all parties to carry out clinical trial to modify the practice of conversion therapy for late stage gastric cancer, and determine the proper selection of suitable candidates for conversion therapy, eventually to offer optimal strategy for originally unresectable gastric cancer patients. Thus, this article focuses on reviewing research progress of conversion therapy for originally unresectable late stage gastric cancer.

OBJECTIVETo evaluate the feasibility and safety of intracorporeal Roux-en-Y esophagojejunostomy via the transoral anvil(OrVil) by mini-laparotomy anastomosis during laparoscopic total gastrectomy (LTG) for gastric cancer.

METHODSFrom March 2010 to December 2016, 414 consecutive gastric adenocarcinoma patients underwent either intracorporeal Roux-en-Y esophagojejunostomy (n=43) via the OrVil or extracorporeal circular anastomosis (n=371) via auxiliary incision during LTG. After generating propensity scores with six covariates, including gender, age, body mass index (BMI), neoadjuvant chemotherapy, tumor location, and tumor size, 43 patients undergoing OrVil method (OrVil group) were matched with 43 patients undergoing extracorporeal circular anastomosis approach (extracorporeal anastomosis group). Operation-associated parameters and safety were compared between the two groups.

RESULTSBoth groups were balanced regarding baseline variables (all P > 0.05). The total operative time [(235.6±49.8) minutes vs. (221.1±46.5) minutes, t=1.397, P=0.166] and anvil insertion time [(10.0±3.2) minutes vs. (10.6±4.5) minutes, t=-0.671, P=0.504] were not significantly different between the two group, whereas the duration of reconstruction and the mean length of minilaparotomy [(48.3±12.0) minutes vs. (55.9±12.3) minutes, t=-2.899, P=0.005; (5.6±0.6) cm vs. (8.1±2.2) cm, t=-7.118, P=0.001] in the OrVil group were significantly shorter. The number of retrieved lymph nodes, mean blood loss and proximal resection margin were not significantly different between two groups (all P > 0.05). As a whole, OrVil group had advantages over extracorporeal anastomosis group during the postoperative recovery course. The time to liquid intake [(3.7±1.8) days vs. (6.2±7.2) days, t=-2.236, P=0.030], time to fluid diet [(4.8±2.3) days vs. (7.2±7.1) days, t=-2.013, P=0.048], and time to semi-fluid diet [(6.7±2.9) days vs. (10.2±9.6) days, t=-2.245, P=0.029] were significantly shorter in the OrVil group. The first ambulatory time, time to first flatus and length of hospital stay were not significantly different between two groups(all P>0.05). The morbidity of intraoperative complication [7.0%(3/43) vs. 4.7%(2/43), χ²=0.000, P=1.000] and postoperative complication [30.2%(13/43) vs. 20.9%(9/43), χ²=1.484, P=0.223], and even the distribution of severity (χ²=0.013, P=0.990) between the two groups were not significantly different. The incidence of anastomotic leakage (AL) was 9.3% (4/43) and 18.6% (8/43) in the OrVil group and extracorporeal anastomosis group respectively without significant difference (χ²=1.550, P=0.213). Multivariate analysis showed that the OrVil anastomosis was not a risk factor of AL(HR=0.663, 95%CI:0.120-3.674, P=0.638).

CONCLUSIONSIntracorporeal esophagojejunostomy using the OrVil system is more minimally invasive and convenient to operate without increasing the risk of operation-related complication. Thus it may be a potential safe approach to optimize the reconstruction for LTG.

Anastomosis, Surgical ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Laparotomy ; Postoperative Complications ; Propensity Score ; Retrospective Studies ; Stomach Neoplasms ; surgery ; Treatment Outcome