Polysaccharide of Atractylodes macrocephala Koidz alleviate lipopolysaccharide-stimulated liver inflammation injury of goslings through miR-223/NLRP3 axis

Abstract

Lipopolysaccharide (LPS) infection could cause severe liver inflammation and lead to liver damage, even death. Previous studies have shown that polysaccharide of Atractylodes macrocephala Koidz (PAMK) could protect liver from inflammation caused by LPS in mice. However, whether PAMK could alleviate liver inflammatory injury in other animals with LPS is still unknown. For evaluating whether PAMK could alleviate liver inflammatory injury in goslings with LPS, a total of 80 healthy 1-day old Magang goslings were randomly divided into 4 groups (control group, PAMK group, LPS group, and PAMK+LPS group). Goslings in control group and LPS group were fed with basal diet, and goslings in PAMK group and PAMK+LPS group were fed basal diet supplemented with 400 mg/kg PAMK to the end of trial. On 24 d of age, goslings in the control group and PAMK group were intraperitoneal injected 0.5 mL normal saline, and goslings in LPS and PAMK+LPS groups were intraperitoneal injected with LPS at 5 mg/kg BW. The serum and liver samples were collected for further analysis after treatment of LPS at 6, 12, 24, and 48 h. Furthermore, the hepatocytes were extracted from goose embryo to measure the expression of the key genes of miR-223/NLRP3 axis. The results showed that PAMK pretreatment could maintain normal cell morphology of liver, alleviate the enhanced levels of biochemical indexes ALT and AST, decrease the levels of IL-1β and IL-18, increase the relative mRNA expression of miR-223, and decrease the expression of NLRP3, Caspase-1, and cleaved Caspase-1 in liver and hepatocytes of goslings induced by LPS. These results indicated that PAMK could relieve inflammatory liver tissue damage after LPS treatment and downregulate the level of inflammation factors via miR-223/NLRP3 axis, thus playing a liver protective role in liver inflammation injury in goslings.

Keywords: gosling; lipopolysaccharide; liver inflammation; miR-223/NLRP3 axis; polysaccharide of Atractylodes macrocephala Koidz.

Figure 1

Result of H.E. staining of goslings liver (H.E. staining, 660 ×). (A–D) control group; (E–H) PAMK group; (I–L) LPS group; (M–P) PAMK + LPS group; the images from left to right show the liver at 6 h, 12 h, 24 h, and 48 h after LPS treatment. Control, LPS group fed a basal diet; PAMK, PAMK+LPS group fed a diet supplemented with PAMK at 400 mg/kg. Abbreviations: HS, hepatic sinusoid; HC, hepatic cords; HV, hepatic venule; triangle, fat vacuole; circle, inflammatory cell infiltration.

Figure 2

Effect of PAMK on the activities of serum ALT and AST in goslings with liver inflammatory injury. Data are expressed as the means ± SD, n = 5. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05). Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Figure 3

Effect of PAMK on the cytokines concentration of IL-1β and IL-18 in hepatic tissue. Data are expressed as the means ± SD, n = 5. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 4

Effects of PAMK on mRNA relative expression of miR-223 and NLRP3 in the hepatic tissue. Data are expressed as the means ± SD, n = 5. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 5

Effect of PAMK on protein relative expression levels of NLRP3, Caspase-1 and cleaved Caspase-1 in the hepatic tissue. (A) Expression of protein relative to GAPDH at 6 h after LPS treatment. (B) Expression of protein relative to GAPDH at 12 h after LPS treatment. (C) Expression of protein relative to GAPDH at 24 h after LPS treatment. (D) Expression of protein relative to GAPDH at 48 h after LPS treatment. Data are expressed as the means ± SD, n = 5. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 6

Effect of PAMK on the concentration of IL-1β and IL-18 in hepatocytes by LPS-stimulated. Data are expressed as the means ± SD, n = 6. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 7

miR-223 could inhibit the expression of NLRP3. (A) Comparison of relative fluorescence values between wild-type and mutant plasmids. (B & D) The efficiency of the miR-223 mimic in hepatocytes was detected by real-time quantitative PCR. (C & G) The efficiency of the miR-223 inhibitor in hepatocytes was detected by real-time quantitative PCR. (E & F) The efficiency of the miR-223 mimic in hepatocytes was detected by western blot. (H & I) The efficiency of the miR-223 inhibitor in hepatocytes was detected by western blot. Data are expressed as the means ± SD, n = 3. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 8

Effect of miR-223 overexpression on the expression of NLRP3 and downstream genes in hepatocytes by LPS-stimulated. Data are expressed as the means ± SD, n = 4. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 9

Effect of PAMK treatment on the expression of NLRP3, Caspase-1 and cleaved Caspase-1 in hepatocytes by LPS-stimulated after miR-223 inhibition. PL: PAMK+LPS group. Data are expressed as the means ± SD, n = 4. Different letters mean significant difference (P < 0.05); the same letter indicates no significant difference (P > 0.05).

Figure 10

Scheme summarizing the protective effects of PAMK on LPS-induced liver injury via the miR-223/NLRP3 axis.