The effect of metformin and mesenchymal stem cells drived exosome on high glucose induced inflammatory responses in HepG2 cells

Abstract

Diabeteses is a highly prevalent metabolic disorder in advanced societies. Insulin resistance is associated with complications such as hyperglycemia, hyperlipidemia, and compensatory hyperinsulinemia. Insulin resistance causes inflammation of the liver, which if left untreated can lead to cirrhosis, fibrosis and even liver cancer. Metformin is the first line of treatment for Diabeteses, which lowers blood sugar and increases insulin sensitivity by inhibiting gluconeogenesis in liver cells. Studies also show that metformin has anti-inflammatory properties. One of the ways to treat hepatitis is to use cell therapy. Among the cells used are mesenchymal stem cells. However, due to the side effects of therapeutic treatment, the exosomes of these cells, which have similar properties, are used in various new studies. Exosomes of mesenchymal stem cells have anti-inflammatory and regulatory properties. Material and Methods: We isolated mesenchymal stem cells from human umbilical cord and confirmed their characteristics by confirmatory tests. Then, serum-free culture medium was used to extract the exosome from these cells, and after 3 days, the supernatant soup of the cells was used to isolate the exosome. Exosome separation was performed by EXOCIB kit using kit protocol. The specificity of the exosomes was confirmed using DLS and TEM methods. In order to induce inflammatory conditions in HepG2 cells, DMEM culture medium with high glucose was used and in fact a model of insulin resistance was developed. Metformin and mesenchymal stem cell-derived exosomes were used to reduce the production of proinflammatory cytokines, and finally the production and expression of IL-6, TNF-α, IL-1β and IL-10 cytokines in the groups The patient was evaluated by ELISA and real time PCR. The rate of apoptosis and necrosis in different groups was also assessed by AnnexinV-PI kit. Result: The results of confirmatory tests showed that the isolated vesicles are in the range of average size of exosomes and in an approximate size of 45 nm. The results of ELISA and real time PCR tests showed that the simultaneous use of exosomes derived from mesenchymal stem cells and metformin had better effects than the use of either alone. Gene expression and production of IL-6, TNF-α and IL-1β cytokines were significantly reduced in the combination therapy groups. Also, the level of gene expression and production of anti-inflammatory cytokine IL-10 in this group was associated with a significant increase.