Changes in animal characteristics and histopathological observations
The body weight of the rats in the CCl4 group was lower than that in the control group after medication administration (Fig. 1). The decreases of body weights of colchicine and RGXJD groups were much greater than that of CCl4 group. At the end of the experiment, absolute and relative liver weights were measured, which were significantly increased in CCl4 intoxicated rats as compared with those in control rats. Meanwhile, these animal characteristics were improved by colchicine and RGXJD groups.
The biochemical characteristics demonstrated that RGXJD could effectively reduce the CCl4 caused injury in rats (Fig. 2A-D). The elevated level of alanine transaminase (ALT) and aspartate aminotransferase (AST) in the CCl4 group indicated that the liver was damaged, releasing ALT and AST into the blood. However, after treatment with colchicine or RGXJD, the activities of ALT, AST were significantly decreased compared with the model group, which indicated that RGXJD has positive effects on CCl4-induced liver fibrosis in rats, and possess potent hepatoprotection.
Histopathological examination revealed the changes of clinical chemistry parameters (Fig. 2E-H). Severe changes in liver morphology were observed in the model group after administration of CCl4, including fiber interval formation, fatty steatosis, and infiltration of inflammatory cells in the portal area, and most rat livers appeared to have pseudo lobules, which demonstrated liver fibrosis was successful established. By contrast, no apparent change was observed in the liver of control rats. Small circular vacuoles indicating slight hepatic steatosis were observed in the liver of RGXJD groups. Furthermore, the colchicine treatment group also displayed similar protective effects.
1H-NMR analysis and metabolic profiling in serum and liver
The representative 1H-NMR spectra of serum and liver tissue samples obtained from all groups were shown in Figs. 3 and 4. The assignments of endogenous metabolite assignments were chemical shifts reported in previous literature.
They reveal different metabolic profiles between the control and treated groups. In total, 19 and 32 metabolites were identified and quantified in serum (Fig. 3) and in liver tissue (Fig. 4), respectively.
The unsupervised principal component analysis (PCA) and supervised PLS-DA models were reconstructed to determine the metabolic changes and to characterize the metabolite profile of the control and RGXJD treated samples. The control and model groups had significant differences in PLS-DA analysis of serum and liver spectrometry, indicating that the metabolic profile changes after liver fibrosis, which are induced by CCl4, while the colchicine and RGXJD ones appeared close to the control group. The OPLS-DA scores plots showed apparent separation between treated groups and control group, with a satisfactory goodness of fit and predictability. At the same time, we observed that there is no difference between the RGXJD and control groups. The corresponding loadings plot combined with the VIP values (VIP > 1.0) from the pattern recognition model, screened out potential biomarkers for the differentiation of colchicine, RGXJD and CCl4 groups. Alternation of potential biomarkers was evaluated by Student’s t test, and the statistical significance was accepted if p < 0.05. The 1H-NMR detected relative integral levels of metabolites indicating differences between all groups are listed in Tables S1 and S2.
For the serum, the coefficient-coded loadings plot (Fig. 5) and Table S1 showed the relevant changes in the endogenous metabolites responsible for the separation comparison of treated groups and control. In addition, the coefficient-coded loadings plot of RGXJD group compared with the model group, the levels of isoleucine, glycine, methanol, alanine increased obviously and had decreased in uridine levels.
For the liver, the coefficient-coded loadings plot (Fig. 6) and Table S2 showed the relevant changes in the endogenous metabolites responsible for the separation comparison of treated groups and control. In addition, the coefficient-coded loadings plot of RGXJD group compared with the model group, the levels of NAD+, xanthine, tyrosine, fumare, inosine, isoleucine, UDP-Glucose, betaine, sarcosine, glutathione, dimethylamine, lactate, leucine increased obviously and had decreased in uridine and valine levels.
Metabolic pathways analysis
In addition, we performed the metabolic pathway analysis of the integrated 27 differential metabolites. Through the MetaboAnalyst 4.0 (www.metaboanalyst.ca) topology analysis, we found that RGXJD exposure disturbed 26 metabolic pathways (Fig. 7). The correlation network was constructed by searching through the Kyoto Encyclopedia of Genes and Genomes KEGG (http://www.kegg.jp) pathway database. In the present study, our results illustrated that the comprehensive metabolic profile changes triggered by CCl4-induced liver injured rats were largely linked to amino acid, purine metabolism, and energy metabolism, and the associated metabolic pathways of each substance are summarized in Fig. 8.
The Metabolic Pathway Analysis in Serum Samples
The 11 metabolic differences were observed in serum, which were Alanine, citrate, citric acid, glycine, inosine, lysine, methanol, uridine, 3-hydroxy butyrate and isoleucine (Fig. 7A). There was 1 metabolic pathways affected (impact > 0.1 and p < 0.05), Glyoxylate and dicarboxylate metabolism.
The Metabolic Pathway Analysis in Liver Samples
The 21 metabolic differences in liver were alanine, glycerin, inosine, inositol, leucine, N-acetyl glycoprotein, phenylalanine, serine, tyrosine, valine, betaine, dimethylamine, fumarate, glutathione, isoleucine, lactate, NAD+, sarcosine, UDP-glucose, uridine and xanthine (Fig. 7B). There were 4 metabolic pathways affected (impact > 0.1 and p < 0.05), including Aminoacyl-tRNA biosynthesis, Phenylalanine, tyrosine and tryptophan biosynthesis, Glycine, serine and threonine metabolism and Phenylalanine metabolism.