Rosavin improves insulin resistance and alleviates hepatic and kidney damage via modulating the cGAS-STING pathway and autophagy signaling in HFD/STZ-induced T2DM animals

Abstract

Background: Inflammation-mediated insulin resistance in type 2 diabetes mellitus (T2DM) increases complications, necessitating investigation of its mechanism to find new safe therapies. This study investigated the effect of rosavin on the autophagy and the cGAS-STING pathway-related signatures (ZBP1, STING1, DDX58, LC3B, TNF-α) and on their epigenetic modifiers (miR-1976 and lncRNA AC074117.2) that were identified from in silico analysis in T2DM animals. Methods: A T2DM rat model was established by combining a high-fat diet (HFD) and streptozotocin (STZ). After four weeks from T2DM induction, HFD/STZ-induced T2DM rats were subdivided into an untreated group (T2DM group) and three treated groups which received 10, 20, or 30 mg per kg of R. rosea daily for 4 weeks. Results: The study found that rosavin can affect the cGAS-STING pathway-related RNA signatures by decreasing the expressions of ZBP1, STING1, DDX58, and miR-1976 while increasing the lncRNA AC074117.2 level in the liver, kidney, and adipose tissues. Rosavin prevented further weight loss, reduced serum insulin and glucose, improved insulin resistance and the lipid panel, and mitigated liver and kidney damage compared to the untreated T2DM group. The treatment also resulted in reduced inflammation levels and improved autophagy manifested by decreased immunostaining of TNF-α and increased immunostaining of LC3B in the liver and kidneys of the treated T2DM rats. Conclusion: Rosavin has shown potential in attenuating T2DM, inhibiting inflammation in the liver and kidneys, and improving metabolic disturbances in a T2DM animal model. The observed effect was linked to the activation of autophagy and suppression of the cGAS-STING pathway.

Fig. 1. A) Volcano plots in the GSE142025 dataset between the analytic groups. For a particular analysis, each point represents a gene, and the blue and red-highlighted data points correspond to down- and up-regulated differentially expressed genes, respectively. Genes with no significance in their expression are observed as black dots. B) Uniform Manifold Approximation and Projection (UMAP) graph showing how samples in each group were related to each other.

Fig. 2. The networks of protein–protein interactions (PPIs) created with the string tool for (A) the cGAS-STING pathway-related genes and (B) key mRNAs and biochemical signatures, (https://stringdb.org; Jan 2022). (C) The way the key RNA and biochemical signatures interact with epigenetic miR-1976 using miRWalk 3.0 (https://mirwalk.umm.uni-heidelberg.de/). (D) Overview of the bioinformatics set up. (E) Flowchart summarizing the experimental animal design and study methodology. 3UTR: 3 untranslated region, 5UTR: 5 untranslated region, Adv DKD: advanced diabetic kidney disease, Ear DKD: early diabetic kidney disease, PPI: protein–protein interaction.

Fig. 3. The effect of rosavin in HFD/STZ-induced T2DM animals. (A) A schematic presentation for animal experimental design. (B) Final body weight. (C) Body records along the experiment. Data are mean ± SD (n = 8 rat). *P < 0.001 vs. CON; ^#P < 0.001 vs. T2DM. ^aP < 0.05 vs. R. rosea-10. Vehicle 1: 0.1 m citrate buffer. Vehicle 2: saline.

Fig. 4. Effect of rosavin on the histology of the liver, kidney, and adipose tissue in T2DM rats. CON group demonstrated normal apparent intact well-organized hepatocytes with intact subcellular details (arrow) with intact hepatic vasculatures (star). Kidney tissue showed abundant records of intact renal tubular segments with intact tubular epithelium (arrow), and intact renal corpuscles (red star). The T2DM group showed microvesicular hepatic steatosis alternated with vacuolar changes all over hepatic lobules (blue arrow), moderate periportal mononuclear inflammatory cell infiltrates (red arrow), and focal hepatocellular necrosis replaced with inflammatory cells (star). The kidney revealed marked vacuolar degenerative changes and nuclear pyknosis (red arrow), significant tubular dilation (star), and moderate interstitial mononuclear inflammatory cell infiltrates (arrowhead). R. rosea-10 group showed minimal insignificant protective efficacy. The kidney showed moderate tubular degenerative changes with occasional focal necrotic segments (red arrow), apparent intact segments (black arrow), and minimal interstitial inflammatory cell infiltrates. R. rosea-20 showed significantly higher hepatoprotective efficacy with abundant apparent intact hepatocytes (black arrow), fewer persistent fatty changes (blue arrow), and minimal abnormal infiltrates with focal necrotic lesions. The kidney showed preserved tubular epithelium integrity (black arrow), moderate persistent tubular dilatation (star), and minimal inflammatory infiltrates. R. rosea-30 group showed the highest protective efficacy with more organized hepatic parenchyma (blue arrow) and higher apparent intact hepatocytes (black arrow). The kidney showed preserved tubular epithelium integrity (black arrow), mild luminal exfoliation of some epithelial cells (arrowhead), and minimal inflammatory infiltrates. Adipocyte cell size increased in the T2DM group but decreased in R. rosea-treated groups in a dose-dependent manner. The data are expressed as means ± SD (n = 6). *P < 0.001 vs. CON; ^#P < 0.001 vs. T2DM.^aP < 0.05 vs. R. rosea-10. ^bP < 0.05 vs. R. rosea-20.

Fig. 5. Showing the effect of rosavin on the expression of RNA molecular signatures in the liver, kidney, and adipose tissue. Values are expressed as mean ± SD; number of rats = 8 rats per group. *P < 0.001 vs. the CON group. ^#P < 0.001 vs. the T2DM group, ^aP < 0.05 vs. R. rosea-10 group. ^bP < 0.05 vs. R. rosea-20 group. One-way ANOVA and Tukey's test.

Fig. 6. Photomicrographs of LC3B and, TNF-α immunohistochemistry-stained liver and kidney sections. LC3B and TNF-α positive-stained cells were identified by their dark brown color.

Fig. 7. Proposed molecular mechanism of rosavin treatment in HFD/STZ-induced T2DM animals.