Role of toll-like receptor 4/mutant myeloid differentiation primary response 88/nuclear factor kappa-B mediated inflammation in diabetes mellitus with Northwest dryness syndrome

Abstract

Objective: To investigate the role of toll-like receptor 4 (TLR4)/mutant myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa-B (NF-κB) signaling pathway-mediated inflammation in diabetes mellitus with Northwest dryness syndrome.

Methods: Rats were randomly divided into the normal control, type 2 diabetes (T2DM) model, Northwest dryness syndrome + T2DM (Northwest dryness), and simple internal dampness + T2DM (internal dampness) groups. Enzyme-linked immunosorbent assay was used to detect biochemical indexes and inflammatory factors. The histopathological observation was performed. Quantitative real-time polymerase chain reaction and Western blot analysis were used to detect the mRNA and protein expression levels, respectively.

Results: Compared with the T2DM group, the glycosylated hemoglobin A1c, insulin, glucose tolerance, the homeostasis model assessment of insulin resistance, tumor necrosis factor-α, interleukin 1β, interleukin 16, malondialdehyde, blood lipid, alanine aminotransferase, and aspartate aminotransferase were significantly elevated in the internal dampness group. Their levels were significantly elevated in the Northwest dryness group than in the T2DM and internal dampness groups. The superoxide dismutase, glutathione peroxidase, liver glycogen, and organ-to-weight ratio were significantly declined in the internal dampness group and the Northwest dryness group than in the T2DM group. However, these levels were elevated in the Northwest dryness group than in the internal dampness group. Moreover, the mRNA expression levels of interferon regulatory factor 5 and NF-κB p65, and the protein expression levels of TLR4, MyD88, and NF-κB were significantly higher in the internal dampness and the Northwest dryness groups than the T2DM group. Additionally, the mRNA and protein levels were significantly higher in the Northwest dryness group than in the internal dampness group.

Conclusion: Northwest dryness syndrome-mediated TLR4/MyD88/NF-κB pathway and chronic inflammation might be associated with the occurrence and development of T2DM.

Keywords: NF-kappa B; Northwest dryness syndrome; diabetes mellitus; inflammation; myeloid differentiation factor 88; toll-like receptor 4.

Figure 1. Pathological changes of pancreas, liver, perirenal adipose and epididymal adipose

A: HE staining detection of the lesions of the pancreas in the four groups (magnification × 400). A1: control group; A2: T2DM group; A3: Northwest dryness group; A4: internal dampness group. B: HE staining detection of the lesions of the liver in the four groups (magnification × 400). B1: control group; B2: T2DM group; B3: Northwest dryness group; B4: internal dampness group.C: HE staining detection of the lesions of the perirenal adipose in the four groups (magnification × 400). C1: control group; C2: T2DM group; C3: Northwest dryness group; C4: internal dampness group. D: HE staining detection of the lesions of the epididymal adipose in the four group (magnification × 400). D1: control group; D2: T2DM group; D3: Northwest dryness group; D4: internal dampness group. E: PAS staining detection of the liver glycogen content in the four groups (magnification × 400). E1: control group; E2: T2DM group; E3: Northwest dryness group; E4: internal dampness group. Control group: rats were not treated; T2DM group: T2DM model was established; Northwest dryness syndrome + T2DM group: Northwest dryness syndrome and T2DM model were established; Internal dampness syndrome + T2DM group: Internal dampness syndrome and T2DM model were established. HE: hematoxylin-eosin; T2DM: type 2 diabetes mellitus.

Figure 2. Changes in expression levels of IL-1β, TNF-α, and IL-16 in serum and liver tissues of rats by ELISA

A: The levels of IL-1β, TNF-α, and IL-16 in serum of rats. A1: analysis of IL-1β expression; A2: analysis of TNF-α expression; A3: analysis of IL-16 expression. B: The levels of IL-1β, TNF-α, and IL-16 in liver tissues of rats. B1: analysis of IL-1β expression; B2: analysis of TNF-α expression; B3: analysis of IL-16 expression. Control group: rats were not treated (n = 6); T2DM group: T2DM model was established (n = 6); Northwest dryness syndrome + T2DM group: Northwest dryness syndrome and T2DM model were established (n = 6); Internal dampness syndrome + T2DM group: Internal dampness syndrome and T2DM model were established (n = 6). TNF-α: tumor necrosis factor-α; IL-1β: Interleukin 1 Beta; IL-6: Interleukin 16; T2DM: type 2 diabetes mellitus; ELISA: enzyme-linked immunosorbent assay. One-way analysis of variance and the least significance difference test were used for analysis. Data were expressed as mean ± standard deviation. Compared with the control group, ^aP<0.05; compared with the T2DM group, ^bP<0.05; and compared with the Northwest dryness group, ^cP<0.05.

Figure 3. Changes in the levels of SOD, MDA, liver glycogen, and GSH-PX in rats

A: SOD level in rats from each group; B: MDA level in rats from each group; C: liver glycogen level in rats from each group; D: GSH-PX level in rats from each group. T2DM: type 2 diabetes mellitus; Control group: rats were not treated (n = 6); T2DM group: T2DM model was established (n = 6); Northwest dryness syndrome + T2DM group: Northwest dryness syndrome and T2DM model were established (n = 6); Internal dampness syndrome + T2DM group: Internal dampness syndrome and T2DM model were established (n = 6). SOD: superoxide dismutase; MDA: malondialdehyde; GSH-PX: glutathione peroxidase. One-way analysis of variance and the least significance difference test were used for analysis. Data were expressed as mean ± standard deviation. Compared with the control group, ^aP < 0.05; compared with the T2DM group, ^bP < 0.05; and compared with the Northwest dryness group, ^cP<0.05.

Figure 4. Changes in the protein expression levels of TLR4, NF-κB p65, and MyD88in the liver, perirenal adipose, and epididymal adipose tissues in rats

A: TLR4, NF-κB p65, and MyD88 protein expression level in the liver tissue by Weston blot. A1: pictures of Western blot analysis of TLR4, NF-κB p65, and MyD88; A2: Western blot analysis of TLR4 expression; A3: Western blot analysis of NF-κB p65 MyD88 expression; A4: Western blot analysis of MyD88NF-κB p65 expression. B: TLR4, NF-κB p65, and MyD88 protein expression level in the perirenal adipose tissue by Weston blot. B1: pictures of Western blot analysis of TLR4, NF-κB p65, and MyD88; B2: Western blot analysis of TLR4 expression; B3: Western blot analysis of NF-κB p65 expression; B4: Western blot analysis of MyD88 expression. C: TLR4, NF-κB p65, and MyD88 protein expression level in the epididymal adipose tissue by Weston blot. C1: pictures of Western blot analysis of TLR4, NF-κB p65, and MyD88by Weston blot; C2: Western blot analysis of TLR4 expression; C3: Western blot analysis of NF-κB p65 expression; C4: Western blot analysis of MyD88 expression. Lane 1: Control; Lane 2: T2DM; Lane 3: northwest dryness syndrome; Lane 4: internal dampness. TLR4: toll-like receptor 4; MyD88: mutant myeloid differentiation primary response 88; NF-κB: nuclear factor kappa-B; T2DM: type 2 diabetes. Control group: rats were not treated (n = 6); T2DM group: T2DM model was established (n = 6); Northwest dryness syndrome + T2DM group: Northwest dryness syndrome and T2DM model were established (n = 6); Internal dampness syndrome + T2DM group: Internal dampness syndrome and T2DM model were established (n = 6). One-way analysis of variance and the least significance difference test were used for analysis. Data were expressed as mean ± standard deviation. Compared with the control group, ^aP<0.05; compared with the T2DM group, ^bP<0.05; and compared with the Northwest dryness group, ^cP<0.05.