AICAR suppresses TNF-α-induced complement factor B in RPE cells Scientific Reports

In order to create a simplified overview, we rated a compound as beneficial when it increased growth, ATP and decreased ROS compared to the values on GAL. The evaluation was designated with a plus sign for each favorable parameter while a negative effect was designated with a minus sign. When no parameter was significantly altered by a compound it was designated non significant (ns). To summarize, AICAR was the most favorable compound with positive effects on several parameters in five out of six patient cells. Although sodium phenylbutyrate slightly increased ROS in some cells, the overall score was positive in fifty percent of the patients (Fig. 2D).

All animal procedures were reviewed and approved by the Animal Ethics Committee of Wenzhou Medical University. The AMPK-mediated suppression of protein synthesis including peptide hormones, growth factors, cytokines and chemokines results in the inhibition of substances contributing to cell growth. AMPK is a heterotrimeric protein that responds to increases in the ratio of adenosine-5′-monophosphate (AMP)-ATP by inducing catabolic processes that produce ATP, and by repressing energy-consuming anabolic mechanisms including protein synthesis 30,31. AICAR has been reportedly used as a pharmacological activator of AMPK in several reports 23,24. A commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems) was used to determine the amounts of IL-8 and GROα (R&D Systems) in the supernatants. The sensitivities of the assays for IL-8 and GROα were 0.7pg/mL and 4.4 pg/mL, respectively.

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• In the present study, we found that these early activation markers are only expressed in 7-AAD- live T cells after activation regardless of AMPK expression.
• Both groups were incubated with 2.0 mM of AICAR starting 1 hour prior to stimulation with TNF-α (10 ng/mL) for 24 hours.
• AICAR has been used clinically for myocardial protection in coronary artery bypass grafting and myocardial ischemic injury (Rao et al., 2016).
• In short, the cells were incubated with 1× fixation buffer for 7 min at room temperature.
• This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).
• A commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems) was used to determine the amounts of IL-8 and GROα (R&D Systems) in the supernatants.

On the other hand, under standard culture condition, AICAR would induce apoptosis through AMPK-mediated JNK activation; while metformin did not induce apoptosis (Fig. 6). Therefore, we speculated that different culture conditions and differences in downstream mediators were the primary causes for the differential regulations of apoptosis by AICAR and metformin in INS-1E cells. This speculation provided a new insight into the current understanding of the controversies regarding the role of AMPK in β-cell apoptosis.

In conclusion, our study shows for the first time the effects of AICAR on complement regulation, abrogating TNF-α-induced CFB expression in RPE cells. However, pharmacologic and genetic evidence demonstrated that AICAR inhibitory effects on TNF-α induced CFB are AMPK-independent. Collectively, this suggests that AICAR could be used as a regulator of CFB, yet further experiments are required to elucidate the AMPK independent anti-inflammatory mechanism of AICAR in complement regulation in the RPE. Levels of cytoplasmic ROS were evaluated by DCFDA at P5 (before incubation with any of the compounds) and P10 (after continuous in vitro culture in the presence of AICAR, NAM, and concomitant AICAR+NAM). Simultaneous use of AICAR and NAM reduced cytoplasmic ROS as there was no significant change in the levels of ROS at P10 in comparison with P5. The untreated cells in the control group and AICAR-treated cells showed a dramatic rise in intracellular ROS.

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Activated EGFR upregulated MUC1-CT expression in EGFR-TL-induced lung tumour tissues. Co-treating patient and transgenic mouse lung-tissue-derived tumour organoids with AICAR and JAK1 and EGFR inhibitors reduced their growth. It has been acknowledged that 5-FU is usually used in combination with other chemotherapeutic drugs to enhance its therapeutic efficacy for CRC patients. In order to examine whether AICAR can sensitize anticancer effect of 5-FU, we evaluated the effect of AICAR on 5-FU-induced apoptosis. We found that the viability of cells treated by AICAR in combination with 5-FU was significantly lower than that of controls (Figure 3A). Flow cytometry showed that the percentage of apoptotic cells was significantly higher in cells treated by AICAR in combination with 5-FU compared to controls (Figure 3B).

Multicellular spheroid growth assay

An adenosine analog that is phosphorylated in whole cells to form 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5’-monophosphate (ZMP), which stimulates AMPK activity. AICAR steroids acts by entering nucleoside pools, significantly increasing levels of adenosine during periods of ATP breakdown. Mimics the effects of insulin on the expression of two gluconeogenic genes PEPCK and glucose-6-phosphatase. Even though previous studies support AICAR’s treatment in leukaemia, hepatocarcinoma, and prostate cancer, our cell-based screening of cytotoxicity of AICAR was limited to its relatively smaller scale in lung cancer.

However, the present raptor-mTOR findings are inconsistent with the decreased eIF4F complex formation, polysome aggregation, and RNA content that we observed in the obese mice. This suggests that an essential initiation factor may not be recruited to the preinitiation complex because of dysregulated TORC1 signaling. One explanation for obese skeletal muscle atrophy may be the recruitment of eukaryotic initiation factors to the preinitiation complex. EIF3 is responsible for providing a scaffold for the recruitment of raptor-mTOR to promote the phosphorylation of S6K1 on T389 and eventual recruitment of the eIF4F complex (22). Since S6K1 was hyperphosphorylated in fasted obese muscle, it is unlikely that eIF3 is responsible for the dysregulation of translation. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) is a substance produced naturally by the body that stimulates AMP activated protein kinase (AMPK), a protein that regulates metabolism in a variety of ways.

Most importantly, Nrf2 gene deletion markedly weakened the protective effects of AICAR to prevent SAP-induced oxidative stress and NLRP3 inflammasome activation in the liver tissues of L-arginine-induced PALI mice (Figure 7F, Figures 8B,C). AMPK can regulate a variety of physiological and pathological effects through multiple pathways to affect cell metabolism and survival (Carling, 2017; Ramirez Reyes et al., 2021). Nonetheless, our findings indicate that activation of Nrf2 by AICAR mediates important roles in ameliorating hepatic oxidative stress and inhibiting NLRP3 inflammasome pathway activation in PALI mice regardless of whether Nrf2 is the master pathway. We explored the hypothesis that the molecular basis of AICAR in improving PALI is attributed to its anti-inflammatory capability. These observations confirm that AICAR treatment protects against PALI in sodium taurocholate-induced SAP rats, likely by inhibiting the inflammatory response in the liver.