There was a higher miR-34a expression in the Ob group. This result is in line with that of Ahmadpour et al. (2018), who found an increase in the miR-34a expression in DIO rats 17. Increased relative expression of miR-34a in the white adipose tissue during obesity has a relationship with adipogenesis. Some miRNAs, along with other adipogenesis master regulators, such as Peroxisome proliferator-activated receptor gamma (PPARγ) and CCAT-enhancer-binging proteins (C/EBP), increase the transcription of adipogenic genes. According to Francisco (2010), miR-34a upregulation is followed by an increase of pre-adipocyte differentiation gene expression, while downregulation of miR-34a decreases the adipogenic gene expression 18.
Increased miR-34a causes chronic inflammation 3. miR-34a plays a role in suppressing the expression of Kruppel-like factor 4 (Klf4), which causes macrophage infiltration3. Macrophage infiltration is associated with an increase in TNF-α, which causes chronic inflammation. TNFα produced by macrophages plays an essential role in the regulation of adipokines in adipocytes. TNF-α induces proinflammatory cytokines through nuclear factor kappa B (NFκB). The binding of TNF-α to its receptor induces the production of proinflammatory cytokines through NFκB-dependent and -independent mechanisms. It also causes the production of proinflammatory cytokines, such as IL-Iβ, and IL-6 19–21.
In this study, we did not measure the inflammatory cytokines in DIO rats. However, we showed the effects of miR-34a expression, as evidenced by decreased Fgfr and Klb expression. This study is in line with the work of Gallego-Escuredo (2015), who reported that obesity leads to the reduced expression of FGFR and beta-klotho receptors in the white adipose tissue 22. Hale (2012) stated that the expression of beta-klotho, Fgfr-1c, and Fgfr2c are downregulated in the adipose tissue of DIO rats 23. Delfin et al. (2012) found decreased expression of Fgfr1 and co-receptor beta-klotho in DIO rats. The decrease in the receptor numbers reduces the amount of binding with Fgf21. This decrease is supported by our previous research, which showed that the levels of Fgf21 expression in white adipose tissue in DIO mice are lower than that of the control group 24.
The decreased expression has an impact on endocrine Fgf21 communication in the adipose tissue and is the beginning of the development of Fgf21 resistance 19. Fgf21 resistance leads to an increase in Fgf21 secretion in the liver 24. The results of previous studies have shown that the expression of Fgf21 in the liver of the DIO rats is higher compared to the normal group. Increased expression levels of Fgf21 in the liver are a result of the disruption of Fgf21 uptake in the white adipose tissue. This happens as a compensation effect due to Fgf21 resistance. The increase in Fgf21 expression in the liver is followed by an increase in circulation 24. Our results are in line with those of Geng et al. (2019), who found that serum Fgf21 levels in the DIO rats are six times higher than that in the normal group 18. Research by Morrice et al. (2017) showed an increase in Fgf21 expression levels in the liver in mice with Fgf21 resistance 25.
The current study showed that the administration of HSE could manage Fgf21 resistance through increased expression of Fgfr and Klb 26. H. sabdariffa is suggested to suppress miR-34a as a regulator of FGFR and beta-klotho expressions 27, 28. However, the mechanism of how HSE downregulates miR-34a has not been proven. Several studies showed the potential of polyphenol compounds in other plant extracts that are microRNA modulators. Baselga-Escudero et al. (2015) showed that proanthocyanin, a component of polyphenols found in grapes and cocoa, downregulates miR-33. It also suppresses miR-122, which inhibits lipogenesis. Meanwhile, polyphenols from HSE modulate miR-122, miR-103, and miR-107 in hyperlipidemic rats 29.
The potential of HSE in suppressing miR-34a has been suggested through PPARγ and C/EBP expression. Additionally, a decrease in PPARγ and C/EBP expression is associated with miR-34a suppression. According to Lavery (2016), there is a decrease in the expression of Pparγ and C/EBP in miR-34a knockout rats 28. According to Kim (2007), HSE suppresses transcription factors PPARγ and CEBP/α 30. Thus, the inhibition of PPARγ and C/EBP as a result of HSE administration has the potential to suppress the expression of miR-34a 31.
However, suppressing the expression of miR-34a through the administration of HSE is related to the dose. Here, we showed that the administration of HSE at a dose of 200 mg/kg in DIO rats did not demonstrate a significant reduction in miR-34a expression compared to the Ob group. In contrast, the administration of HSE at a dose of 400 mg/kgBW was found to significantly reduce the expression of miR-34 compared to that of the Ob group. Even though miR-34 expression levels do not reach normal levels, beta-klotho and FGFR expression can still increase 32, reaching normal levels of FGFR1 expression.
The increased expression of beta-klotho and FGFR can also be influenced by HSE, which directly suppresses chronic inflammation. Some studies have found that polyphenols in the HSE can inhibit proinflammation by suppressing NFκB. Zeng et al. (2016) found that polyphenols increase the expression of Fgfr1 and beta-klotho in rats fed with a high-fat diet 33. This is because polyphenols act as anti-inflammatory agents by decreasing NFκB expression. The results of Gamboa-Gomez (2015) indicate that HSE significantly reduces TNFα induced by NFκB 34. In addition, the anthocyanins, namely cyanidin and delphinidin, in the HSE, also reduce TNFα expression. That is because anthocyanin inhibits the activation of NFκB by inhibiting the degradation of IκB and inhibiting the activation of IκB kinase, thereby preventing the phosphorylation of NFκB 19. In the current study, we did not measure NFκB expression levels; thus, we could not directly prove that the increase in Klb and Fgfr expression was associated with a decrease in NFκB. This study proves the potential of HSE in increasing the expression of Fgfr and Klb, such that it can manage Fgf21 resistance in DIO rats 35.
Our previous research showed that there is a higher than normal increase in Fgf21 in the adipose tissue of DIO mice administered HSE at a dose of 400 mg/kgBW. The management of FGF21 resistance is indicated by the activation of the FGF21 signaling pathway in the adipose tissue. FGF21 binds with its receptor to activate signaling via PGC1α, which is a transcription coactivator that controls energy metabolism. The present study showed an increase in Ppargc1a expression in the Ob-Hib400 group.
SIRT1 stimulates increased PGC1α activity. After FGF21 binds to FGFR and beta-klotho, it induces browning of white adipose tissue to beige adipose tissue through SIRT1 activation, which results in PGC1α deacetylation to induce UCP-1 expression 5. The results of the current study also showed an increase in Ucp-1 expression after HSE administration. The rise in Ucp-1 expression was shown not only in the Ob-Hib400 group but also in the Ob-Hib200 group.
The increase in UCP-1 expression is not only affected by PGC1α activation but also by other factors 33. UCP-1 expression is influenced by several paths that are regulated by major transcription factors, such as PPARγ and PRDM16 36.
PPARγ is a crucial transcription factor in the differentiation of brown and white adipocytes. PPARγ is needed for the recruitment of PRDM16 to the PPARγ transcription complex, which achieves the browning process 37. Thus, HSE not only activates the PGC1α pathway but is also thought to play a role in the PPARγ and PRDM16 pathways. However, further research is needed to prove this. According to Tian (2017), polyphenol content from green tea improves transcriptional regulators, such as PPARγ, PGC1α, PRDM16, and UCP1, for the browning process 38.