Mesenteric Lymph Duct Drainage Attenuates Lung Inflammatory Injury and Inhibits Endothelial Cell Apoptosis in Septic Rats

Abstract

The present study was to investigate the effect of mesenteric lymph duct drainage on lung inflammatory response, histological alteration, and endothelial cell apoptosis in septic rats. Animals were randomly assigned into four groups: control, sham surgery, sepsis, and sepsis plus mesenteric lymph drainage. We used the colon ascendens stent peritonitis (CASP) procedure to induce the septic model in rats, and mesenteric lymph drainage was performed with a polyethylene (PE) catheter inserted into mesenteric lymphatic. The animals were sacrificed at the end of CASP in 6 h. The mRNA expression levels of inflammatory mediators were measured by qPCR, and the histologic damage were evaluated by the pathological score method. It was found that mesenteric lymph drainage significantly reduced the expression of TNF-α, IL-1β, and IL-6 mRNA in the lung. Pulmonary interstitial edema and infiltration of inflammatory cells were alleviated by mesenteric lymph drainage. Moreover, increased mRNA levels of TNF-α, IL-1β, IL-6 mRNA, and apoptotic rate were observed in PMVECs treated with septic lymph. These results indicate that mesenteric lymph duct drainage significantly attenuated lung inflammatory injury by decreasing the expression of pivotal inflammatory mediators and inhibiting endothelial apoptosis to preserve the pulmonary barrier function in septic rats.

Figure 1

Morphologic changes of lung and evaluation of lung injury under light microscopy (magnification, ×100). There was no evidence of lung injury in the control group (a) and sham surgery group (b). In contrast, evidence of increased interstitial edema and inflammatory dell infiltration was found in the sepsis group (c). The injury degree of interstitial edema and inflammatory cell infiltration was ameliorated in the sepsis+MLD group (d). The data of the lung injury score are expressed as mean ± SE, n = 8 (e). Results were compared by one-way ANOVA with Student-Newman-Keul's posthoc test. ^∗p < 0.05vs. control group; ^#p < 0.05 vs. sepsis group.

Figure 2

Morphologic changes of intestinal mucosa and evaluation of gut injury with Chiu's scores under light microscopy (magnification, ×200). The control group (a) and the sham operation group (b) had normal villi and glands. By contrast, severe edemas of mucosal villi accompanied with severe intestinal gland injury were observed in the sepsis group. In addition, a large number of intestinal villi disintegrated, the gap of epithelial cells increased, and severe hemorrhage was present, indicative of severe mucosal damage in the sepsis group (c). The putrescence and desquamation of epithelial cells in the intestinal mucosa were attenuated, but mucosal sloughing could be seen at villi tips, and the gap between epithelial cells increased slightly in the sepsis+MLD group (d). The data of Chiu's scores were expressed as mean ± SE, n = 8 (e). Results were compared by one-way ANOVA with Student-Newman-Keul's posthoc test. ^∗p < 0.05 vs. control group.

Figure 3

Morphologic changes of liver and evaluation of liver injury under light microscopy (magnification, ×200). There was no apparent change of liver in the control group (a) and sham surgery group (b). In the sepsis group, swollen hepatocyte, raritas, or lucency kytoplasm were observed. In additional, dilated sinus hepaticus and lymphocyte infiltration in converged tube were also observed in the sepsis group (c). The injury degree of sinus hepaticus dilation and inflammatory cell infiltration was improved in the sepsis+MLD group (d). The data of the liver injury score are expressed as mean ± SE, n = 8 (e). Results were compared by one-way ANOVA with Student-Newman-Keul's posthoc test. ^∗p < 0.05 vs. control group.

Figure 4

Expression of TNF-α, IL-1β, and IL-6 mRNA in intestine, liver, and lung tissues. Data were expressed as mean ± SD (n = 8). ^∗p < 0.05 vs. control group, ^#p < 0.05, sepsis+BTED group vs. sepsis group.

Figure 5

Effect of mesenteric lymph drainage on MPO levels of intestine, liver, and lung in septic rats. Results are presented as mean ± SD (n = 8).^∗p < .01 vs. control group.

Figure 6

The morphological characteristics of PMVECs treated with mesenteric lymph (magnification, ×400). There was no morphologic change in PMVECs cocultivated with 10%PBS (a). When cocultured with 10% septic lymph, PMVECs showed shrinkage and detached from the bottom of the culture flask (c). In contrast, the injury of PMVECs was significant lessened when cocultured with 10% normal lymph (b). The cell viability of PMVECs was decreased by treatment with septic lymph for 24 h compared with that in the lymph group as measured by the MTT assay (d). TNF-α, IL-1β, and IL-6 mRNA expressions were significantly increased in PMVECs cocultivated with septic lymph compared with that in the normal lymph group (e). Results were analyzed using two-way repeated measures ANOVA with protected posthoc testing. ^∗p < .01 vs.10% PBS group and 10% normal lymph.

Figure 7

Flow cytometric analysis of PMVEC apoptosis using Annexin V-FITC/PI staining. The apoptosis rates of PMVECs were increased when treated with either septic lymph or normal lymph, compare with the control group. (a) Cocultivation of PMVECs with 10% PBS. (b) Cocultivation of PMVECs with 10% normal lymph. (c) Cocultivation of PMVECs with 10% septic lymph. (d) Data are presented as mean ± SD (n = 3), ^∗p < .01 vs.10% PBS group and 10% normal lymph.

Figure 8

Effect of septic mesenteric lymph duct drainage on pulmonary endothelial apoptosis. Number of TUNEL-positive endothelial cells per 5 fields (hpf) in rats subjected to sham surgery, CASP sepsis model, and CASP+MLD. Data are shown as mean values ± SD (n = 8 per group). ^∗p < .01 vs. control and sham surgery groups. ^#p < .01 vs. sepsis group.