3.1 Erucamide effects on pathological changes in the liver tissue of rats with Stress-induced liver injury
The pathological changes of liver tissue were inspected under a light microscopy. The results demonstrated that liver cells were actually arranged with distinct nucleoli and involved plentiful cytoplasm with a red color and round nuclei. The nuclear chromatin appeared blue. No unusual pathological changes in liver tissues in the Normal group were inspected (Fig. 2A). In the SLI group, liver cells were obviously injured, showing distinct liver cell swelling, with aberrant nucleus size, hepatic cord mistake, and large area of vesicular degeneration of liver cells (Fig. 2B). In the SLI + Era group, cells with hydropic degeneration were inspected in liver tissues,but a lesser extent than in the SLI group, and liver cells were mildly dropsical (Fig. 2C)
3.2 Effects of erucamide on AST and ALT in serum of rats with Stress-induced liver injury
Compared with those in the Normal group, serum ALT and AST activities of rats in the SLI group were obviously increased (P < 0. 001). In contrast to those in the SLI group, serum AST activities of rats in the SLI + Era groups were unsignificantly reduced, and ALT activities of rats in the SLI + Era groups were obviously reduced (P < 0. 001), see Fig. 3.
3.3 Effects of erucamide on AST and ALT in liver tissues of rats with Stress-induced liver injury
AST and ALT activities of liver tissues in the SLI group significantly distinct from those in the Normal group (P < 0.01).In contrast to those in the SLI group, AST and ALT activity was significantly reduced (P < 0.05) in the SLI + Era group, as shown in Fig. 4.
3.4 Screening of differentially expressed genes by mRNA expression chip
The consequence of distinctively expressed genes screened by mRNA expression microarrays illustrated that there were 5432 distinctively expressed genes in the Normal group contrast to the SLI group, among which the expression of 3139 (57.79%) genes was obviously up-regulated, and the expression of 2293(42.21%) genes was obviously down-regulated (see Fig. 5A). In contrast to the SLI group, there were 319 distinctively expressed genes in the SLI + Era group,among which the expression of 132 (41.38%) genes was obviously up-regulated, and the expression of 187(58.62%) genes was obviously down-regulated (see Fig. 5B).
3.5 Functional enrichment analysis of differentially expressed gene KEGG
From metabolic analysis, the mechanism of erucamide protectingrats from Stress-induced liver injury is mainly related to homeostasis, endocrine metabolism and neurosecretory metabolism, among which the regulation of carbon metabolism and amino acid metabolism is the main potential mechanism(see Fig. 6 and Fig. 7).
From the analysis of the organism's organic system, the mechanism of erucamide protecting rats from Stress-induced liver injury is mainly related to the immune system, endocrine system and nervous system(see Fig. 6 and Fig. 7).
From the analysis of human diseases, the mechanism of erucamide protecting rats from Stress-induced liver injury is highly related to immune diseases, metabolic diseases and neurological diseases(see Fig. 6 and Fig. 7).
From the results of the KEGG enriched carbon metabolism pathway analysis, it can be seen that compared with the Normal group, the catabolism of glycine was increased in the SLI group increased. On the contrary, in contrast to the SLI group, the SLI + Era group reduced the metabolism of glycine(see Fig. 8 and Fig. 9:Black frame). In the central nervous system, glycine is an inhibitory neurotransmitter. Glycine and glutamate are both agonists in the central nervous system, regulating the nervous system and related neurological diseases. Therefore, erucamide may prevent stress liver injury by inhibiting the cleavage of glycine.
3.6 Quantitative real-time RT-PCR
The DLD, AMT, UNC5B, GPSM3 and IGSF11 genes were choosed for real-time quantitative PCR. ( Fig. 10 ). The consequence demonstrated that the DLD, AMT, UNC5B, GPSM3 and IGSF11 genes played an important part in the Stress-induced rats liver injury. Among them, DLD and AMT are the key enzymes of the glycine cleavage system. In contrast to the Norma group, the gene expression of DLD and AMT in the SLI group has a significant increase ( ###P < 0.001 ), and Erucamide can inhibit its expression. In addition, the main biological function of UNC5B gene is to participate in cell apoptosis [26]. Compared with the SLI group, Erucamide can inhibit its expression, indicating that erucamide can inhibit cell apoptosis. In addition, the abnormal expression of GPSM3 [27] and IGSF11(an immunoglobulin (Ig) superfamily member) [28] genes is associated with inflammation. Experimental results clarified that Erucamide can also regulate inflammation.
3.7 Assessment of beneficial effects of erucamide and glycine in protecting Crot-induced LO2 cell damage
In this study, in contrast to the Normal group,Crot (300uM) treatment significantly decreased the viability of LO2 cell (P < 0.001). On contrary༌erucamide at 2.5µM、5µM、10 µM concentrations also prevented Crot-induced LO2 cell damage, and erucamide at 10 µM concentrations significantly prevented Crot-induced LO2 cell damage(P < 0.001,see Fig. 11A). In addition, glycineat 1.5mM、3mM、6 mM concentrations also prevented Crot-induced LO2 cell damage ; and glycine at 6 mM concentrations Significantly prevented Crot-induced LO2 cell damage(P < 0.05, see Fig. 11B). Overall, these data suggested that Erucamide and Glycine were effective in preventing Crot-induced LO2 cell damage.
3.8 Effects of erucamide on AST and ALT in LO2 cell with Crot-induced LO2 cell damage
AST and ALT activities in the Cort group obviously differed from those in the Normal group (P < 0.01). In contrast to those in the Cort group, AST activity was obviously reduced (P < 0.05) in the erucamide group, as shown in Fig. 12.
3.9 Effects of glycine on AST and ALT in LO2 cell with Crot-induced LO2 cell damage
AST and ALT activities of LO2 cell in the Cort group obviously differed from those in the Normal group (P < 0.01,P < 0.05). In contrast to those in the Cort group, AST and ALT activity was obviously reduced (P < 0.001༌P < 0.05) in the glycine group, as shown in Fig. 13
3.10 Hoechst 33342 staining to observe the results of cell apoptosis
The chromatin distribution of LO2 cells in the normal group was uniformly diffused low-intensity fluorescence (see Fig. 14A), and normal organelles and intact cell membranes were visible. After adding 300 µmol/L corticosterone for 24 h, a large number of nuclei appeared densely stained with dense condensed morphology or granular fluorescence (see Fig. 14B), indicating that the action of 300 µmol/L corticosterone for 24 h caused obvious apoptosis of LO2 cells. Compared with corticosterone model group, glycine (see Fig. 14C) and erucamide (See Fig. 14D) The drug can reduce the apoptosis induced by corticosterone.