SLS reduced loss of weight and DAI of model mice
The time-related change of body weight of mice was shown in Fig1A. There was gradual downregulation of body weight in DSS group compared with Ctrl group, which was consisted with previous studies. DSS+SD group exhibited greater loss of body weight than DSS group. However, SLS reduced the loss of weight UC mice with spleen-deficiency. The time-related change of DAI was showed in Fig 1B. In comparison with Ctrl group, DAI in DSS and DSS+SD group both increased, while DAI in SLS group decreased significantly in comparison with the DSS+SD group. It was suggested that spleen-deficiency might exacerbate the macroscopic manifestations in mice intervened by DSS, and SLS could relieve the manifestations in mice of UC with spleen-deficiency.
Length of colon in different groups of mice
The comparation of colon length was shown in Fig1C and D. Compared with Ctrl group, the colon length in DSS and DSS+SD group both reduced significantly and it was even shorter in DSS+SD group, while the mice in SLS group exhibited longer colon than that in DSS+SD group. It was suggested that DSS shorten the colons of mice successfully and spleen-deficiency exacerbated this alteration, however, SLS treatment ameliorated the shortening of colon in mice of UC with spleen-deficiency.
Spleen and thymus weight in different groups of mice
As shown in Fig 2, the spleen weight and spleen index increased in the DSS group and decreased in the DSS+SD group compared with the Ctrl group. After intervention with SLS, both the spleen weight and spleen index increased significantly compared with DSS+SD group. Compared with the Ctrl group, both thymus weight and thymus index decreased significantly in DSS and DSS+SD group, however those in SLS group exhibited significant elevation compared with DSS+SD group. It was suggested the intestinal inflammatory induced by DSS upregulated the spleen weight and downregulate the thymus weight in mice, while spleen-deficiency both downregulated the spleen and thymus weight.
Histopathological observation of colon tissue in different groups of mice
The histopathological alteration in different groups was observed by H&E staining. As Fig 3 showed, there was complete colonic epithelial structure and normal crypt morphology in colon tissue of Ctrl group. However, colon tissue of DSS and DSS+SD group were damaged. The histopathological alteration of DSS group were charactered as destroyed colonic epithelial structure, incomplete crypt morphology and inflammatory infiltration in mucosal layer, and that of DSS+SD group were charactered as completely destroyed colonic epithelial structure, disappeared crypt morphology and a large amount of inflammatory infiltration in the mucosal layer and submucosa layer. After intervention with SLS, the colon tissue was shown as relatively complete colonic epithelial structure and crypt morphology and inflammatory infiltration in the mucosal layer. It was indicated that spleen-deficiency aggravated histopathological damage of colon tissue induced by DSS and SLS ameliorated the damage at a microscopic level.
The level of inflammatory cytokines in different groups
In comparison with the Ctrl group, the relative mRNA expression of IL-1β, TNF-ɑ and IL-6 in DSS and DSS+SD group increased significantly and those in DSS+SD group was higher. After SLS treatment, lower mRNA expression of inflammatory cytokines was shown than DSS+SD group (Fig 4), which signified spleen-deficiency promoted the intestinal inflammation in UC mice and SLS reduced the intestinal inflammation.
The relative intensity of serum metabolites in different groups of mice
The result of sPLS-DA analysis showed there were differences in metabolites among the four groups (Fig 5A), and the heatmap verified the difference (Fig 5B). In comparison with Ctrl group, DSS+SD group exhibited decreased relative intensity in PC(O-7:0/O-7:0), Phosphocholine, PC(O-18:1(1E)/0:0), PC (14:0/0:0), LysoSM(d18:0), LysoPC(20:0), Docosatrienoic Acid and increased relative intensity in LysoPC(22:6) and Glycerophospho-N-Palmitoyl Ethanolamine. After intervention with SLS, the mice showed consistent tendency with Ctrl group (Fig 5D-L) and it was indicated that UC mice with spleen-deficiency suffered from imbalance of metabolites, while SLS had an effect on regulating the metabolites balance.
The intestinal barrier in different groups of mice
As shown in the AB-PAS staining results, colonic goblet cells and mucus decreased in DSS and DSS+SD group, while SLS treatment relieved the reduction of goblet cells and restored the mucus. As shown in the IHC results, the expression of ZO-1 and Muc-2 decreased compared with Ctrl group, while SLS treatment upregulated the expression of ZO-1 and Muc-2 compared with DSS+SD group (Fig 6). It was indicated that spleen-deficiency exacerbated the damage of tight junction and mucosal barrier in colon and SLS ameliorated the damage effectively.
The expression of SIRT1、PGC1ɑ、TFAM mRNA in different groups of mice
The relative mRNA expression of SIRT1, PGC1ɑ and TFAM were detected by RT-PCR as shown in Fig 7. Compared with Ctrl group, both the DSS and DSS+SD group exhibited significant reduction in the relative mRNA expression and DSS+SD group exhibited further reduction. Compared with DSS+SD group, the relative mRNA expression of SIRT1, PGC1ɑ and TFAM increased significantly in SLS group. The results signified UC mice existed mitochondrial biogenesis dysfunction and spleen deficiency might aggravate this dysfunction. Besides, SLS could promote mitochondrial biogenesis in UC mice with spleen-deficiency through SIRT1/PGC-1ɑ pathway.
The protein expression of SIRT1、PGC1ɑ、TFAM in different groups of mice
The relative protein expression of SIRT1、PGC1ɑ、TFAM were detected by western blot as shown in Fig 7. The relative expression of above proteins performed the same trend as mRNA expression. Compared with Ctrl group, DSS and DSS+SD group exhibited significant reduction in the relative protein expression and DSS+SD group exhibited further reduction. After SLS intervention, the relative protein expression of SIRT1、PGC1ɑ、TFAM in mice upregulated significantly in comparison with DSS+SD group.
As shown in above results, spleen-deficiency was considered as an unfavorable factor to mitochondrial biogenesis in UC mice and SLS could be acted as an effective intervention to promote mitochondrial biogenesis through SIRT1/PGC-1ɑ pathway.
The activity of Na+-K+-ATPase and Ca+-Mg+-ATPase in different groups of mice
Compared with Ctrl group, Na+-K+-ATPase and Ca+-Mg+-ATPase activity in DSS and DSS+SD group reduced significantly (P < 0.05, P < 0.01) and those in DSS+SD group showed further reduction, while the ATPase activity in SLS group performed upregulation compared with DSS+SD group. (Fig 8A and B) The results suggested there was cell transport dysfunction in UC mice and spleen-deficiency aggravated the dysfunction and SLS was suggested could improve cell transport and mitochondrial function in UC mice with spleen-deficiency.
The copy number of mitochondrial DNA in different groups of mice
Compared with the Ctrl group, the copy number of mitochondrial DNA (mtDNA) in DSS and DSS+SD group were significantly reduced (P < 0.05, P < 0.01) and the DSS+SD group showed further reduction. However, the copy number of mitochondrial DNA in SLS group exhibited significant elevation compared with DSS+SD groups after SLS treatment. (Fig 8C) The results suggested there was mitochondrial transcription dysfunction in UC mice and spleen-deficiency exacerbated the dysfunction and SLS was considered as an effective measure to improve mitochondrial DNA transcription in UC mice with spleen-deficiency.