| Objective: This study was designed to compare the differences in biological characterization and morphological changes of hypothalamus and colon tissues between the rat model of abnormal sapra syndrome formed by combined stimulation of multiple factors and the rat model of ulcerative colitis(UC) induced by TNBS-ethanol enema; and the differences of metabolic components as well as transcriptional profile in hypothalamus and colon between the two rat models, to further analyze the function of thyroid hormone in inflammation and overall metabolism changes, and to explore the nature of abnormal sapra syndrome and the material basis as well as the molecular basis of UC induced by abnormal sapra syndrome. Methods: Multi-factors, including dry and hot feeding environment, hot and spicy feeds(black pepper, ginger, and saffron), clipping tails, and noise, were used to establish a rat model of abnormal sapra syndrome. And TNBS and ethanol enema were applied to induce rat UC model. Biological characterization and morphological changes of the rats were observed. Changes in metabolites in hypothalamus and colons of the rats with abnormal sapra syndrome induced by multiple factors and the TNBS-UC rats were detected by using the nuclear magnetic resonance(NMR). Transcript profile in hypothalamus and colon tissues of both abnormal sapra syndrome rats and TNBS-UC rats were comprehensively analyzed using RNA-seq to screen the differentially expressed genes. Secondly, function clustering of differentially expressed genes was performed with the GO method, and differentially expressed genes-related signaling pathways were enriched with the KEGG. Thirdly, the protein interaction network was constructed, and the interaction between the differentially expressed genes was analyzed to screen the important differentially expressed genes. And fourthly, expression levels of these differentially expressed genes were verified by real-time fluorescence quantitative PCR. Seven serum indicators of thyroid function and biochemical indexes related to serum glucose and lipid metabolism were detected, and the relationships between thyroid hormone levels and metabolic components in rats of the abnormal sapra syndrome and the UC group were preliminarily determined by correlation analysis. Meanwhile, based on the results of the serum metabonomics, the global network regulation chart was plotted. Results: 1) Rat model that simulated the clinical manifestation of abnormal sapra syndrome was successfully established by multi-factor compound modeling method, and the TNBS-UC model was also created. For the abnormal sapra syndrome group, visual observation showedminor bleeding in colon tissue of the animals, and microscopic observation after HE staining revealed a few inflammatory cells in the colon tissue in the rats of the same group. Cerebral vascular dilatation and congestion was observed under the microscope in the hypothalamus of the abnormal sapra syndrome rats, with widened perivascular space and some round nerve cells. For the animals in the UC group, necrosis, congestion, and edema were found on the intestinal wall of the colon, with membranous substance attached on the surface of some deep and large ulcers. Congestion and clear edema in peripheral intestinal mucosa were seen, and the intestinal walls of the lesions were markedly thickened with stiffness. The degeneration and necrosis of the epithelial cells in intestines mucous were found by the microscope after HE staining, with massive neutrophil infiltration as well as cellulose exudation and patchy hemorrhages in mucosal lamina and submucosal layers. This was consistent with the pathological changes of acute inflammation and the changes of acute phase in human UC. Microscope results showed cerebral vascular dilatation and congestion in the hypothalamus of the UC rats, with widened perivascular space and some round nerve cells. 2) Results of hypothalamic metabolism showed that metabolites of significantly high abundance included valine, leucine, isoleucine, ethanol, 4-amino butyric acid, N-acetyl aspartic acid, choline, and inositol, and those with significantly low abundance were glutamate and taurine in the abnormal sapra syndrome group. And the metabolites of significantly high abundance in the UC group were ethanol, γ-aminobutyric acid, N-acetyl aspartate, lactic acid, and phenylalanine. It was found by RNA-Seq analysis of samples that most pathways in hypothalamus of the rats with abnormal sapra syndrome were associated with neural signaling pathways, such as neuroactive ligand-receptor interaction, cocaine addiction, amphetamine addiction, serotonergic synapse, and dopaminergic synapse. While for the rats in the UC group, the up-regulated genes in the thalamus tissues were enriched in the adipocytokine signaling pathway, the calcium signaling pathway, the tyrosine metabolism, the cytokine-cytokine receptor interaction, and the PI3K-Akt signaling pathway. Co-expression analysis of differential genes in the abnormal sapra syndrome group and the UC group was performed, and two pathways related to the thyroid hormone response were found, which were the thyroid hormone synthesis and the autoimmune thyroid disease pathways, and the differentially expressed genes were Cga and Tshb. In combination with the GO-protein network map and genetic pathway network map, four genes were found to be up-regulated jointly, which were Pmch, Cga, Tshb, and Hcrt.3) Analysis of colonic metabolism showed that the metabolites with significantly high abundance in the abnormal sapra syndrome group included 3-hydroxybutyric acid, lactic acid, succinate, and ATP/ADP, and those with significantly low abundance were GSH, glycerol phosphate metabolite choline, and inositol. The metabolism analysis of the UC group indicated that the metabolites which were significantly increased compared with the normal control group in colon tissues were butyric acid, propionic acid, ethanol, 3-hydroxybutyric acid, acetic acid, succinic acid, methylamine, dimethylamine, propanedioic acid, glucose, and glycogen, and those were significantly decreased in the UC group included alanine, glutamine, glutathione, glycerophosphate choline, inositol, creatine, taurine, and creatinine. The differentially expressed genes obtained in colon tissues were analyzed with GO and KEGG, and it was found that the genes associated with inflammation and immune pathways were down-regulated in the colon of the abnormal sapra syndrome group, while these corresponding genes were up-regulated in the UC group. The union of the genes in the two groups was taken and analyzed by using WGCNA. Modules with low expression in the normal group and the abnormal sapra syndrome group as well as with high expression in the UC group were selected, for which KEGG pathway analysis was performed. Most of the pathways obtained were associated with immune pathways, such as the cytokine-cytokine receptor interaction, the TNF signaling pathway, the PI3K-Akt signaling pathway, and the NF-kappa B signaling pathway. Meanwhile, two pathways related to the thyroid hormone were also identified. Then the main thyroid hormone signaling pathways were screened and it was found through genetic interaction network diagram that Tnf was the core molecule. The co-expression of Thrsp gene revealed that the key enzymes PCK1 and FBP were down-regulated and GK was up-regulated in gluconeogenesis. And at the same time, PKLR and GPD1 in the glycolytic pathway were down regulated, and the expressions of fatty acyl Co A dehydrogenase(Acadsb) gene in fatty acid beta oxidation, however, were up-regulated. In the phospholipid metabolism, the key enzymes phospholipase A and phospholipase C were both down regulated and glutathione S transferase and glutamate cysteine ligase were down regulated.4) Serum levels of T3, T4, FT3, and FT4 in the abnormal sapra syndrome group and the UC group were decreased. The decreases in the abnormal sapra syndrome group were clear, but still in the normal range, while that in the UC group was significantly lower than the normal level. The levels of serum glucose, fructose amine, triglyceride, apolipoprotein B in the UC group were significantly increased, and that of the high density lipoprotein was significantly reduced(P<0.05). Thyroid hormone was negatively correlated with glucose, triglyceride, and total cholesterol, respectively, and was positively correlated with high density cholesterol. Thirteen differential metabolites were ultimately identified in the abnormal sapra syndrome group, and the significantly increased metabolites in the abnormal sapra syndrome included isoleucine, valine, alanine, proline, pyruvate, carnitine, β-hydroxybutyric acid, lactic acid, and creatine, and those significantly reduced metabolites were VLDL, citrulline, and tyrosine. Compared with the normal group, 13 metabolites with differential abundance were found in the UC group, in which those were significantly increased were LDL, lactate, and creatine, and those were significantly decreased were leucine, valine, tyrosine, citrulline, alanine, carnitine, citric acid,-glucose, α-glucose, and methylhistidine. Conclusions: 1) Multi-factors, including hot and spicy feeds, dry and hot feeding environment, clipping tails, noise, and chronic plantar electric stimulation, are used to establish rat model, and excitement, fighting prone, yellow tongue, yellow urine, dry and hard feces, weight loss, increased water drinking and diet appear in the rats. The animal model with syndrome of clinical manifestation of human abnormal sapra is successfully simulated. There is a small amount of inflammatory cell infiltration in this rat model. Ulcerative inflammation in a rat model is reproduced with the induction of TNBS-ethanol enema, and changes of colon tissues appeared are similar to that of the human UC at the acute phase. 2) Abnormal hypothalamic neurotransmitter secretion, disturbed membrane stability, and abnormal energy metabolism are the common manifestations of the animals in both the abnormal sapra syndrome group and the UC group. Upregulated expressions of Cga and tshb genes lead to the increased TSH in the hypothalamus, which may be the response to the stimulation in both groups. They play an important role in the formation of abnormal sapra syndrome and UC, and may be an important molecular basis for the UC induced by abnormal sapra syndrome. 3) The energy imbalance, oxidative damage, and cell membrane instability of colonic tissues are the main factors leading to the occurrence of ulcerative colitis as well as the main causes of UC induced by abnormal sapra syndrome. TNF-α causes oxidative damage in colon cells by inhibiting the activity of GSH, and as a cytokine, it also induce the inflammation through DAG-PKC-NF-κB signal transduction pathway via the dual roles of activation and amplification, and plays an important role in the formation of UC. Thrsp is a key molecule in thyroid hormone metabolism regulation. 4) The decrease of the thyroid hormone content is the common material basis of the abnormal sapra syndrome group and the UC group. The forming process of the abnormal sapra syndrome and the UC, the neuroendocrine system and immune system interacte each other, on the one hand, the immune system through the cytokines(TNF) acts on the nervous- endocrine system, make the abnormal nerve- endocrine hormone release(lower thyroid hormone levels), on the other hand on thyroid hormone acts on whole organism, make the metabolic changes, such as inhibition of glucose metabolism, promote lipolysis, make energy imbalance, and systemic immune disorder and colon lesions. The abnormal sapra syndrome group of serum thyroid hormone levels is low but still in the normal range, means the abnormal sapra syndrome in the sub-health state, the main reasons for it is also one of the reasons of the abnormal sapra syndrome induced UC. |
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