Abstract:Lipopolysaccharide (LPS), a major component of Gram-negative bacterial cell walls, is commonly used as an inducer of intestinal inflammation in animals, but research on its effects in aquatic animals remains limited. This study focused on blackrock fish, Sebastes schlegelii, an important mariculture species in Shandong Province, using intraperitoneal LPS injection to establish an enteritis model. Evaluation included histopathology, immunoenzymatic activity, tight junction proteins, and inflammatory factor gene expression. The control group received sterile phosphate-buffered saline (PBS), while experimental groups were given low-dose (5 mg/kg LPS), medium-dose (10 mg/kg LPS), and high-dose (15 mg/kg LPS). Each group consisted of three replicates, each with 30 fish (initial body weight 85.3±1.7 g). Samples were collected at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h post-injection. The results showed that LPS at 5–15 mg/kg effectively induced intestinal structural lesions, inflammatory responses, and oxidative stress, with severity positively correlated to dose. Examination results indicated that the incidence of intestinal damage was 70% in the low-dose group and reached 100% in both the medium-dose and high-dose groups. Histopathological observations revealed intact intestinal structure in controls, whereas LPS groups showed dose-dependent lesions, primarily inflammatory cell infiltration, villi breakage, lysis, and detachment. In the low-dose group, some fish displayed severe villi structural damage, occasional epithelium damage, intact lamina propria, and infiltration of inflammatory cells into the lamina propria and submucosa. In the medium-dose and high-dose groups, fracturing and detachment were observed. The damage to the epithelium and lamina propria was intensified, and infiltration of inflammatory cells was more pronounced. Notably, the high-dose group showed evident villi detachment and a significant reduction in goblet cell lysis. Antioxidant enzyme assay showed that after LPS stress, SOD activity in the high-dose group was significantly reduced versus the control group at 6 h (P＜0.05). Additionally, SOD activity in the medium-dose group was significantly lower than the control group at 12 h (P＜0.05), and in the low-dose group at 24 h (P＜0.05). The overall trend of SOD activity in all experimental groups decreased then increased, remaining significantly lower than that of the control group's at 24 h post- stress (P＜0.05). MDA activity differed highly significant between the high-dose group and control group from 12 h post-injection (P＜0.001), and the low-dose group differed at 12 h and 24 h (P＜0.05), but no significant differences remained at 96 h. ACP activity in all experimental groups was significantly lower than the control group at 6 h (P＜0.05). It reached its lowest point at 12 h in the medium-dose and 24 h in the high-dose group, with no significant difference by72 h (P＞0.05). AKP activity in the high-dose group was significantly lower than the control group at 6 h (P＜0.05), and in all experimental groups at 12 h (P＜0.05). AKP activity in the low-dose and medium-dose groups recovered by 48 h, showing no significant difference from the control group. LPS injection altered the expression of inflammation-related genes and tight junction protein genes to varying degrees. The overall level of IL-1β gene expression increased then decreased; specifically, in the high-dose group it was significantly higher than the control group at 6 h (P＜0.05). Additionally, the intestinal expression level of IL-8 in all experimental groups was significantly elevated versus the control group at 6 h (P＜0.05), remaining significantly higher in the high-dose group at 96 h (P＜0.001). The IL-10 gene expression level in the high-dose group was significantly lower than the control group at 6 h (P＜0.001), and decreased in the low-dose and medium-dose group at 12 h (P＜0.05). After LPS stimulation, NF-κB expression showed an increasing-and-decreasing trend; it was significantly higher in the high-dose group versus the control at 6 h (P＜0.05), and in the low-dose and medium-dose groups at 12 h, 24 h, and 48 h (P＜0.05). In the high-dose group, the expression of occludin and ZO1 genes was significantly down regulated at 6 h (P＜0.05), although not at 96 h. In the medium-dose group, their expression was significantly lower at 12 h and 24 h (P＜0.001). The relative expression of the ZO1 gene in the low-dose group was significantly lower than that in the control group at 24 h (P＜0.05), with no statistically significant differences at other time points. These gene expression changes indicate that LPS can induce an inflammatory response in S. schlegelii. This study demonstrated that LPS induces intestinal tissue damage, compromises antioxidant capacity, and causes abnormal expression of inflammatory-related genes in S. schlegelii. Consequently, LPS functions as a reliable inducer for establishing intestinal inflammation models in S. 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.
