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IntroductionColon cancer is a kind of highly malignant tumor in digestive tract disease, with the incidence increasing year by year . Since the early clinical symptoms of colon cancer patients are no obvious in the early stage, most cases develop to the middle and late stages when seeking medical treatment, thus worsening the prognoses of the patients . Currently, surgery along with radiotherapy and chemotherapeutic gene or targeted drug therapy is effective in treating colon cancer and increasing the survival time of patients . Despite the multiple treatment options for colon cancer, surgery remains the mainstay [4, 5]. At present, the main surgical methods include traditional open surgery and laparoscopic surgery [6, 7]. Due to the high trauma and complications of traditional open surgery, laparoscopic surgery is preferred by patients .Surgical trauma leads to rapid activation of innate immune cells, including monocytes/macrophages and neutrophils, which release proinflammatory cytokines . Surgical trauma can lead to the suppression of immune function of the body, which immensely increases the risk of bacterial infection . With the advancements in surgical instruments and the minimally invasive technology, laparoscopic surgery is being applied more widely. The influence of laparoscopic surgery on the normal physiological internal environment of the body is mainly manifested in the aspects of internal nerve division, metabolism, immunity and circulation, among which the influence on the stress response of the body is the basis of the change of pathophysiological function . Previous studies have shown that compared to open surgery, laparoscopic surgery has a minor effect on cellular immunity, humoral immunity and non-specific immune system . Recently, cytokine-related cell signal transduction pathways have been widely investigated and attracted curiosity . Studies on mononuclear phagocytes in animal models have determined a minor effect of laparoscopic surgery on cell signaling pathways and an association with the inflammatory response .Herein, colon cancer patients were analyzed to compare the effects of surgical trauma on immune function by assessing the degree of activation degree of cell inflammatory cytokines levels after open and laparoscopic surgery.Materials and methodsInclusion of patients107 patients with colon cancer who were admitted to Affiliated Hospital of Hebei Engineering University were included in the study subjects. Using the random number table method, patients were divided into two groups, the control group (n=54) and the study group (n=53). This study was reviewed and approved by the Ethics Committee of Affiliated Hospital of Hebei Engineering University (approval number: HEU2020081103). All patients were aware of the study design and voluntarily signed informed consent.All patients underwent all preoperative examinations after admission to the hospital, including endoscopy, CT, chest X-ray, histopathology, etc., to evaluate the function of various organs, tumor status, and nutritional status of the patients. Patients with poor nutritional status should be given active nutritional support. All patients began to fast on the afternoon of 1 day before surgery, while taking compound polyethylene glycol electrolyte. Patients were given antibiotics 30 min before surgery to prevent infection. In order to ensure the fairness of the operation, the surgery was accomplished by the same team of doctors. Inclusion criteria: patients were confirmed by colonoscopy biopsy as colon cancer; patients were treated for the first time; imaging examination suggested that surgical resection was feasible; all patients tolerated surgery. Exclusion criteria: patients with abnormal function of other organs; patients prone to other organs or distal metastasis; patients with a history of abdominal surgery; patients with serious underlying medical conditions, such as diabetes complications, or poor blood sugar control; suffering from mental illness and unable to cooperate in this study.Therapeutic schedulePatients in the control group underwent open surgery. The patients were given general anesthesia with endotracheal intubation and placed in the supine position. The rectus abdominis or midabdominal line at the left or right margin was selected as the incision, with a length of 12–20 cm. After opening the skin tissue layer by layer, the blood vessels on both sides of the mesentery and colon were separated. Lymph nodes around the tumor were dissected according to the location of the tumor, and lymph nodes at the root of the tumor and surrounding tissues were completely resected. Next, the submesenteric vessels were incised and clamped, finally the surgical method was selected according to the patient’s condition and tumor location.Patients in the study group underwent laparoscopic complete mesenteric resection surgery, and the anesthesia method was the same as those in the control group. The main operating hole was located at the lower edge of the umbilicus, and the observation hole was located at the McBurney point. The left lower costal margin was used as the auxiliary operation hole. In order to determine whether the tumor has peritoneal metastasis or implantation, patient’s pelvic cavity was detected. The mesangium of the sigmoid colon was incised at the sacral promontory, and the duodenum and pancreatic head were separated along the Toldt’s space, fully exposing the mesenteric artery root and lymph nodes around the tumor were dissected. Then the mesenteric and peripheral omental ligaments were excised, and the bowel was dissociated from the right posterior wall of the abdomen for anastomosis of the stump. For cases with left colon tumors, the submesenteric vessels are first exposed, then an incision was made at the retroperitoneum, 1 cm below the bifurcation of the abdominal aorta, to complete the exposure of the middle colic artery. The inferior mesenteric artery (IMA) trunk was dissected and lymph nodes around the IMA were dissected. After the left colic artery (LCA) and the first branch of sigmoid artery (SA-1) were isolated, the LCA and SA1 were cut off at the root. The mesangium and bowel were cut 10 cm away from each side of the tumor, and end-to-side triangular anastomosis or end-to-side anastomosis was performed. Both groups were given antibiotic anti-infection therapy after the operation.Observation indicatorsPerioperative indexes were collected, including operation time, intraoperative blood loss, anal exhaust, time, postoperative drainage volume and hospital stay. Patients’ complications after surgery were also recorded.Measurement of cytokine levelsA total of 5 mL of peripheral blood was collected from each participant before, after and one day after surgery. Among them, 50 μL blood was collected and added to PMI1640 medium containing 450 μL LPS (100 ng/mL) and stimulated by water bath at 37 °C for 4 h. After centrifugation at 3,000 rpm for 15 min, serum samples were collected and stored in the refrigerator at −20 °C for future use.The frozen serum was melted at a room temperature, and the concentrations of cytokines were determined according to the instructions of the enzyme linked immunosorbent assay (ELISA) kits, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), toll-like receptor 4 (TLR4), cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2). The ELISA kits were purchased from Sangon Biotech (Shanghai, China). In brief, standard products and samples were added into the enzyme label plate, and then washed and stained after incubation. The absorbance (OD) value of the reaction hole sample was measured at 450 nm, and the concentration of each factor in the serum sample was calculated by drawing a standard curve.Analysis of dataAnalysis of the data was proceeded in SPSS 26.0, and figures were drawn in GraphPad 7.0 software. Data were expressed as number or mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Chi-square test were used to judge the difference of variables between groups. p<0.05 indicates a statistically significant difference.ResultsBasic information of the study subjectsOne hundred and seven cases with colon cancer were recruited, including 54 cases underwent open surgery and 53 cases who received laparoscopic surgery. The basic information about the patients is presented in Table 1. Cases in the study and control groups were age and gender-matched (p>0.05). Besides, no significant difference was observed in the course of the disease, tumor location, and TNM stage (p>0.05).Table 1:Basic clinical information statistics of the subjects.ItemsLaparoscopic surgery n=53Open surgery n=54p-ValueAge, years62.06 ± 2.7162.37 ± 2.980.570Sex, n0.631 Male2629 Female2725Course of the disease, years1.24 ± 0.621.27 ± 0.660.794Location (colon)0.772 Left2628 Right2726TNM stage0.784 I, Ⅱ3335 Ⅲ2019Annotation: all data were presented as mean ± standard deviation (SD) or number (n).Effects of different surgery on inflammatory response and TLR-mediated innate immune responsesAs displayed in Figure 1A–C, postoperative CRP, TNF-α and IL-6 levels increased significantly in comparison with pre-operative levels in both groups (p<0.01). One the first postoperative day, CRP and IL-6 levels in both groups decreased significantly, but TNF-α remained at high levels in both groups. CRP, TNF-α and IL-6 levels in the open surgery group were higher than that in the laparoscopic surgery group at the same time point (Figure 1D–F, p<0.01).Figure 1:Inflammatory cytokines levels in two groups. (A–C). Levels of CRP (A), TNF-α (B) and IL-6 (C) before, after and one day after surgery in open surgery and laparoscopic surgery groups. (D–F). Comparison of CRP (D), TNF-α (E) and IL-6 (F) levels between the open surgery group and laparoscopic surgery group at different time points. **p<0.01; ***p<0.001 compared with preoperative group; ###p<0.001 compared with laparoscopic surgery group.Figure 2A showed the changes in the serum CD14 levels. The results demonstrated that there was no significant difference in the CD14 level before and after the laparoscopic surgery. In the open surgery group, the level of CD14 decreased significantly postoperatively and recovered on the first day postoperatively. Postoperative CD14 levels were significantly lower in the open surgery group than in the laparoscopic group (Figure 2D). In addition, serum TLR4 and MD-2 levels decreased significantly in both group after surgery, but recovered from one day after the operation (Figure 2B–C). But in comparison with the laparoscopic surgery group, the postoperative serum TLR4 and MD-2 levels of the open surgery group were significantly lower (Figure 2E–F, p<0.01).Figure 2:Levels of TLR-related cytokines in the two study groups. (A–C). Expression changes of serum CD14 (A), TLR4 (B) and MD-2 (C) before, after and one day after surgery. (D–F). Comparison of CD14 (D), TLR4 (E) and MD-2 (F) levels between the open surgery group and laparoscopic surgery group at different time points. **p<0.01; ***p<0.001 compared with preoperative group; ###p<0.001 compared with laparoscopic surgery group.Furthermore, the blood of the two groups was further stimulated by LPS to mimic the physiological state in vivo, and the levels of inflammatory factors were detected. The results showed that the levels of IL-6 and TNF-α showed no significant change pre- and post-laparoscopic surgery. The levels of IL-6 and TNF-α in the open surgery group were significantly lower than those in the laparoscopic surgery group at all postoperative time points (Figure 3).Figure 3:Serum inflammatory cytokines levels after stimulation by LPS in the two study groups. (A–B) serum TNF-α (A) and IL-6 levels (B) at different time points in the two study groups. (C–D). Serum TNF-α (C) and IL-6 (D) levels between open surgery group and laparoscopic surgery group at different time points. **p<0.01; ***p<0.001 compared with preoperative group; ##p<0.01, ###p<0.001 compared with laparoscopic surgery group.The perioperative performance and postoperative complications of the two groupsAs shown in Table 2, the intraoperative bleeding, postoperative drainage volume of the laparoscopic surgery group was less than those in the open surgery group, whereas the surgery time was longer in the laparoscopic surgery than in the open surgery group (p<0.01). The hospital stay time in the laparoscopic surgery group was shorter than that in the open surgery group, although the difference did not reach the significant level (p>0.05). Nevertheless, the anal exhaust time exhibited no remarkable difference between the two groups (p>0.05).Table 2:Comparison of perioperative indicators between the two groups.IndicatorsLaparoscopic surgeryOpen surgeryp-ValueDuration of surgery157.85 ± 17.65146.31 ± 25.910.008Intraoperative bleeding86.38 ± 15.85220.48 ± 32.85<0.001Postoperative drainage200.74 ± 44.09301.77 ± 74.65<0.001Anal exhaust time3.12 ± 0.793.28 ± 0.730.287Length of stay13.92 ± 4.7915.75 ± 6.180.090Annotation: all data were presented as mean ± standard deviation (SD).Furthermore, the incidence of postoperative complications is shown in Table 3. The common postoperative complications in patients included incision infections, lung and urinary system infection, and anastomotic leak. Of note, cardiovascular and respiratory complications were less in laparoscopic surgery than in open surgery, but no significant difference was detected. Similarly, incision infection and anastomotic leak were also compared between the two groups, but the differences still did not reach the significant levels, although the number seems more in open group than in laparoscopic surgery. Overall, the total number of complications was higher in the open surgery group (p<0.01).Table 3:Comparison of total incidence of complications between the two groups of patients.Site of infectionLaparoscopic surgeryOpen surgeryp-ValueIncision010.320Anastomotic leak130.317Lung020.157Urinary system120.569Total28<0.001Annotation: all data were presented as number (n).DiscussionColon cancer commonly occurs in middle-aged and old men with high morbidity and mortality . The susceptibility of colon cancer is closely related to dietary habits, diet composition, lifestyle, and population aging . Surgical intervention is the main treatment for colon cancer . Traditional open surgery requires a large incision to be placed and also induces significant trauma. Furthermore, it also has the risk of incision-related complications, resulting in poor long-term results .Complete mesenterectomy, a newer approach to treating colorectal cancer, is a simple operation with a short postoperative recovery time, which greatly reduces the mortality rate . The operation under laparoscopy provides an expanded surgical field of view, and reduces the intraoperative damage, which is beneficia for the prognosis and recovery of patients .Studies have confirmed that the immune function and cytokine levels postoperatively are closely related to the degree of surgical trauma, and CRP, IL-6 and TNF-α are assumed to be important indicators for the same . Post-traumatic IL-6 and TNF-α are the main cytokines that induce the synthesis of acute reactive protein in hepatocytes, among which CRP is the most important acute reactive protein . Serum IL-6, TNF-α and CRP levels are positively correlated with the degree of surgical trauma . In the current study, serum CRP, IL-6 and TNF-α levels in both groups were significantly increased at all postoperative time points compared with preoperative levels. One the first day postoperative day, the levels remained at a high level, and the difference was significant in comparison with preoperative levels. However, CRP, IL-6 and TNF-α levels in the laparoscopic group were lower than those in the open surgery group at all postoperative time points. It was concluded that, patients who underwent laparoscopic surgery had a mild inflammatory reaction, and minor cytokines were secreted by immune cells, which further confirmed that laparoscopic surgery caused less body damage and had slight effect on cellular immune function. Moreover, the blood of the two groups was further stimulated by LPS to mimic the physiological state in vivo, and the levels of inflammatory factors were compared. The results showed that the levels of IL-6 and TNF-α showed no significant change pre- and post-laparoscopic surgery. The levels of IL-6 and TNF-α in the open surgery group were significantly lower than those in the laparoscopic surgery group at all postoperative time points. The results illustrated that open surgery weakened the defense function of immune cell defense, while laparoscopic surgery retained the function of immune cells to a certain extent.Severe trauma can damage the barrier of the gastrointestinal mucosal, thus contributing to the release of endotoxins into the blood, even causing sepsis . Sepsis and trauma itself can lead to infection, activation of mononuclear macrophages and excessive release of inflammatory mediators, which may eventually lead to systemic inflammatory response syndrome (SIRS) or (multiple organ dysfunction syndrome (MODS) . LPS is known to be a specific pathogen-associated molecular model that mediates the development of sepsis . Toll-like receptor 4 (TLR4), a member of Toll-like receptor family, is a pattern recognition receptor of LPS . CD14 is a surface differentiation antigen of mononuclear macrophages and a receptor protein of LPS/LPS-binding protein (LBP) complex, which can improve the sensitivity of immune cells to endotoxin . LPS/LBP complex binds to TLR4 transmembrane receptors on the cell membrane, it can transfer stimulus signals from the extracellular to the intracellular. Then mononuclear macrophages are activated, accompanied with the excessive synthesizing and release of TNF-α, IL-6 and other cytokines, finally causes sepsis . MD-2 is a co-receptor that regulates TLR-4 to transduct LPS stimulus signals, which can enhance the body’s response to LPS and promote the expression of TLR4 gene . In conclusion, TLR4, CD14 and MD-2 form LPS recognition receptor complexes with high affinity and signaling function, which jointly mediate the recognition of LPS . In light of the present results, no significant difference was detected in the CD14 level before and after the laparoscopic surgery group. In the open surgery group, the level of CD14 decreased significantly after the operation and recovered on the first postoperative day. Postoperative CD14 levels were significantly lower in the open surgery group than in the laparoscopic group. Ikushima et al. have found that the level of CD14 in monocytes decreased after surgery and trauma, which was related to the degree of trauma, the evidence was consistent with the present findings . In addition, similar trends were also detected in terms of TLR4 and MD-2 between the two groups. In conclusion, open surgery significantly reduced the expression of CD14/TLR4/MD-2, and weakened the function of the body’s immune system, while laparoscopic surgery inflicted minor damage to monocytes and retained their cellular defense function.In this study, the perioperative performance and postoperative complications of the two groups were further analyzed and compared. Notably, few cardiovascular and respiratory complications were observed in cases received laparoscopic surgery, but no significant difference was detected between the two groups. Similarly, incision infection and anastomotic leak were not significantly different between the two groups, although the number seems larger in open group than in laparoscopic surgery. Overall, the total number of complications was larger in the open surgery group, the reason may be that laparoscopic operation causes less damage to the body, it has fewer postoperative complications, and has high safety and low mortality, adding support to the safety and benefits of laparoscopic surgery . But the results of this paper could not clearly reflect the specific type of complications with higher incidence. The small sample size might be an important limitation, and further studies with larger population should be performed to verify the present findings. Moreover, complete mesenteric resection was just performed under laparoscopic surgery, which may have introduced bias, that might be a limitation to compare the therapeutic effect between the two groups and complete mesenteric resection performed under open surgery should be included in future studies. Besides, the long-term oncological outcomes are being evaluated and will be addressed in future studies.In conclusion, the cellular immunity may be better presented after laparoscopic surgery compared to open surgery. Laparoscopic complete mesenteric resection surgery is effective for the treatment of colon cancer.
ONCOLOGIE – de Gruyter
Published: May 1, 2023
Keywords: colon cancer; immune response; laparoscopic complete mesenteric resection surgery; open surgery
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