Abstract:Lipopolysaccharide (LPS), a major component of Gram-negative bacterial cell walls, is commonly utilized as an inducer of intestinal inflammation in animals. However, research on its effects in aquatic animals remains limited. The present study focused on black rockfish (Sebastes schlegelii), an important mariculture fish along the coastal areas of Shandong Province, using intraperitoneal injection of LPS solution as a model to induce enteritis. The evaluation method included multiple parameters, such as histopathology analysis, immunoenzymatic activity, tight junction proteins, and inflammatory factor gene expression levels. The control group received an injection of sterile phosphate-buffered saline (PBS), whereas the experimental groups consisted of the low-dose group (5 mg/kg﹒bw LPS ), the medium-dose group (10 mg/kg﹒bw LPS), and the high-dose group (15 mg/kg﹒bw LPS). Each group consisted of three replicates, with 30 fish (initial body weight 85.3±1.7 g) allocated to each replicate. The samples were collected at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h following intraperitoneal injection of LPS solution. The results indicated that intraperitoneal injection of LPS at doses of 5~15 mg/kg﹒bw effectively induced intestinal structural lesions, leading to inflammatory responses and oxidative stress. The severity of the intestinal inflammatory response in experimental fish was found to be positively correlated with the LPS injection dose. Examination results indicated that the incidence of intestinal damage was 70% low-dose group and reached 100% in both groups medium-dose and high-dose group. Histopathological observations revealed that the intestinal tissue of fish in control group maintained structural integrity, with no apparent signs of intestinal damage, villi breakage, or lysis. In contrast, the intestinal tissue sections of fish in each experimental group injected with LPS exhibited varying degrees of lesions, primarily characterized by inflammatory cell infiltration, villi breakage, lysis, and detachment. The severity of intestinal lesions was positively correlated with the dose of LPS-induced stress. In low-dose group, some individual fish displayed severe structural damage of the intestinal villi, occasional epithelium damage, intact lamina propria, and infiltration of inflammatory cells into the lamina propria and submucosa. In medium-dose and high-dose group, the structural integrity of intestinal villi was significantly compromised, with intestinal villi fracturing and detachment. The damage to the epithelium and lamina propria was further intensified, with a more pronounced infiltration of inflammatory cells observed. Notably, in high-dose group, the detachment of intestinal villi became evident, accompanied by a significant reduction in goblet cell lysis. The results of antioxidant enzyme activity assay showed that, following LPS-induced stress, SOD activity in high-dose group was significantly reduced compared to control group at 6 h (P<0.05). Additionally, SOD activity in medium-dose group was significantly lower than control group at 12 h (P<0.05). SOD activity in low-dose group was significantly decreased relative to control group at 24 h (P<0.05). The overall trend of SOD activity in all experimental groups was decrease followed by an increase. Despite variations in the time to reach the nadir of SOD activity among different experimental groups, SOD activity in all experimental groups remained significantly lower than that in control group at 24 h post-LPS stress (P<0.05). The difference in MDA activity between of high-dose group and control group washighly significant (P<0.001) at 12 h post-injection. The MDA activity of medium-dose group was statistically different from that of control group strating at 12 h after LPS-induced stress (P<0.05). Additionally, MDA activity of low-dose group showed significant difference compared to the control group at both 12 h and 24 h (P<0.05). However, no statistically significant difference in MDA activity were observed between any of the experimental groups and control group at 96 h. At 6 h post-injection, ACP activity in all experimental groups was significantly lower than that in control group (P<0.05). In medium-dose and high-dose group, ACP activity reached its lowest point at 12 h and 24 h, respectively. By 72 h, there was no longer a statistically significant difference between each experimental group and control group (P>0.05). At 6 h after LPS-induced stress, only AKP activity in high-dose group was significantly lower than that in control group(P<0.05). At 12 h post-LPS stress, AKP activity in all experimental groups was significantly lower than that in control group (P<0.05). AKP activity in low-dose and medium-dose group recovered by 48 h, with no significant difference between low-dosegroup, medium-dose group, and control group. The expression of inflammation-related genes and tight junction protein genes in the intestinal tissue of the experimental fish was altered to varying degrees after LPS injection. The overall level of IL-1β gene expression in all experimental groups exhibited an initial increase followed by a decrease. Specifically, after 6 h of LPS stress, the intestinal expression level of IL-1β gene in high-dose group was significantly higher than that in control group(P<0.05). Additionally, the intestinal expression level of IL-8 gene in all experimental groups was significantly elevated compared to control group after 6 h of LPS stress (P<0.05).The intestinal expression level of the IL-8 gene in all experimental groups was significantly higher than that in control group at 6 h post-LPS stress (P<0.05). Furthermore, the intestinal IL-8 expression level in high-dose group remained significantly higher than that in control group after 96 h (P<0.001). Additionally, the intestinal IL-8 expression level in all experimental groups after 6 h of LPS stress was still significantly higher than that in control groupat 96 h (P<0.001). Regarding the IL-10 gene, its expression level in high-dose group was significantly lower thancontrol group at 6 h post LPS stress (P <0.001). Moreover, the intestinal IL-10 expression level gene in low-dose and medium-dose group decreased significantly at 12 h post-LPS stress compared to congtrol group (P<0.05). After LPS stimulation, the expression level of NF-κB, an inflammatory pathway gene, in the fish intestine from all experimental groups showed an increasing-and-decreasing trend compared with that in control group. Specifically, the relative expression level of NF-κB in high-dose group was significantly higher than in control group at 6 h of post-LPS stress (P<0.05). Moreover, the relative expression level of NF-κB gene in low-dose and medium-dose group were significantly higher than that in control group at 12 h, 24 h and 48 h (P<0.05). In high-dose group, the expression of occludin and ZO1 genes was significantly downregulated compared with control group at 6 h (P<0.05), while no statistically significant difference was observed between the expression of occludin and ZO1 genes in high-dose and control group at 96 h. In medium-dose group, the expression of occludin and ZO1 genes was significantly lower than that in control group at 12 h and 24 h (P<0.001). The relative expression of ZO1 gene in low-dose group was significantly lower than that in control group at 24 h (P<0.05), and there was no statistically significant difference in the expression of occludin and ZO1 genes between low-dose group and control group at other time points. These changes in the relative expression of inflammatory genes, signal transduction genes, and tight junction protein genes indicated that LPS can induce an inflammatory response in Sebastes schlegelii. This study has demonstrated that LPS can induce damage to the intestinal tissue structure, compromise antioxidant capacity, and lead to abnormal expression of inflammatory-related genes in Sebastes schlegelii. Consequently, LPS functions as a reliable inducer for establishing intestinal inflammation models in Sebastes schlegelii, thereby providing a robust foundation for further in-depth investigation into the pathogenesis of bacterial intestinal inflammation in marine fish and the efficient screening of preventive and therapeutic drugs.
