Objective. To observe the impact of quercetin and isoquercitrin on gluconeogenesis in hepatocytes. Methods. Mouse primary hepatocytes were cultured with lactic acid and pyruvic acid. After treatment with quercetin and isoquercitrin for 24 hours, the glucose concentration in the culture supernatant was determined. RT-PCR was used to detect the mRNAs of PEPCK, G6Pase, LKB1, and AMPKα. Protein levels of LKB1, AMPKα, and Thr172 phosphorylation were evaluated by Western blot. Results. The glucose concentration in the gluconeogenesis group (GN) was significantly higher than in the control group (C), but the glucose concentrations in the high level quercetin(group 80Q) and high level isoquercitrin (group 80I) were significantly lower than in the group GN, P<0.01. In the group 80Q, and group 80I, the mRNA levels of PEPCK and LKB1were significantly lower than in the group GN (P<0.01), and the G6Pase mRNA were significantly lower than in the group GN (P<0.05). The protein levels of LKB1 and the phosphorylation of AMPKα Thr172 in the group 80Q, group 40I, and group 80I were higher than in the group GN. The effects of quercetin and isoquercitrin on LKB1 and AMPKα were similar to those of metformin. Conclusions. Quercetin and isoquercitrin inhibit gluconeogenesis in hepatocytes, which may be related to the LKB1 upregulation and phosphorylation of AMPKα.
BACKGROUND Anal gland infection is one of the main pathogenic factors of anal fistula. The anal gland is mainly consists of columnar epithelial cells and goblet cells. Goblet cells could secrete mucins which maintain the surface mucous gel layer of intestine to prevent columnar epithelial cells from the direct acting of bacterias. The absence or diminished secretion of goblet cells could lead to the exposure of columnar epithelial cells to bacterias. In recent years, studies have found that most of the microorganisms in perianal abscess and anal fistula are intestinal bacteria. So it can be considered that the occurrence and development of anal fistula is closely related to gut microbiota. However, the molecular mechanism of gut microbiota acting on the epithelial cells of anal gland leading to the occurrence and development of anal fistula has not been explored. METHODS Anal fistula tissues were collected from 30 patients, and HE staining was employed to observe pathological changes of anal gland. Stool specimens were collected from normal group (14 normal subjects), preoperative group (21 subjects before surgery) and postoperative group (16 subjects after surgery). High-throughput 16S rRNA gene sequencing was performed to explore differences among different groups. RESULTS It was found by HE staining that normal anal gland is composed of a large number of columnar epithelial cells as well as goblet cells, while the anal gland of patients with anal fistula is seriously lack of goblet cells. In the normal ones, a large number of vacuoles formed by the dye dissolution of mucin particles can be seen at the top of the goblet cells, and a large amount of mucin secretion can be seen in the center of the anal gland. The results of high-throughput 16S rRNA gene sequencing showed that there were significant statistical differences in species richness, species evenness and species composition between the preoperation group and the normal group (P1 = 0.00004), and between the preoperative group and the postoperative group (P2 = 0.00003). It also showed that PWY-6471 metabolic pathway was the most significant difference between the preoperative group and the postoperative group (P = 0.0033). CONCLUSIONS The occurrence of anal fistula may be related to the abnormal permeability of mucus layer and the change of intestinal flora composition. The absence or diminished secretion of goblet cells may cause exception of the permeability of mucus layer which makes the columnar epithelial cells of anal gland exposed to the direct contact of peptidoglycan. The metabolite of pathogenic microorganisms could activate the signal pathway (i.g. NLR/NF-κB) which leading to the overexpression of inflammatory factors and cause anal fistula.
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