3.6 Isorhamnetin reversed AngII-induced atrial fibrosis and related gene expression in AF model mice
Histological analysis was performed to confirm the inhibitory effect of isorhamnetin on atrial fibrosis. Images obtained from Masson trichome staining showed more disarray fibrosis in the AngII group (Fig. 6A). Furthermore, the quantitative ratio of the fibrotic area to the total area in the AngII group was significantly higher than that in the control group. However, isorhamnetin markedly suppressed AngII-induced atrial fibrosis (Fig. 6B). Tissue fibrosis normally involves the activation of the TGF-β pathway, which promotes collagen deposition. As a result, in the left atrium, two weeks of AngII infusion increased the gene expression of Tgfβ andCol1a1 (collagen type I alpha 1) compared to the controls. However, isorhamnetin diminished the expression of these genes (Fig. 6C-D). These results demonstrate that isorhamnetin has inhibitory effects on fibrosis in the atria.
3.7 Effect of isorhamnetinon AF-related protein expression in atrial tissues
The pathogenesis of AF is a complex process involving not only Ca2+ channels, but also a variety of other molecules (Nattel et al., 2020). CaMKII oxidation usually contributes to diastolic SR Ca2+ leakage and Ca2+ loading (Wang et al., 2018). Cav1.2, a subunit of the L-type voltage-gated calcium channel, is encoded by the calcium voltage-gated channel subunit alpha1 C (CACNA1C) and is involved in the formation of AP and the regulation of blood pressure. TRPC3 and TRPC6 are closely related to AF by promoting myofibroblast formation (Rose et al., 2012). In addition, JNK, ERK, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activate oxidation, hypertrophy, fibrosis, and inflammation (Nattel et al., 2020). The mRNA expression level of Camkii  was not significantly increased in the AngII group compared to the control group (Fig. 7.1A). On the other hand, the mRNA expression levels of Cacna1c  and Ryr2  were increased in the AngII group and decreased in the AngII +isorhamnetin group (Fig. 7.1B-C). The results of western blotting for Ca2+-handling-related molecules and TRPCs are shown in Fig. 7.1D. The protein expression levels of ox-CaMKII and p-RyR2 at Ser 2814 were significantly increased in the AngII group and were significantly decreased in the AngII +isorhamnetin group (Fig. 7.1E-F). The elevated expression levels of TRPC3/6 and CACNA1C induced by AngII were also decreased in the AngII +isorhamnetin group (Fig. 7.1G-I). Finally, the results of western blotting for the structural remodeling-related signaling pathways are shown in Fig. 7.2A. The protein expression levels of p-ERK, p-JNK, and p-NF-κB were remarkably enhanced in the AngII group and notably diminished in the AngII+isorhamnetin groups (Fig. 7.2B-D). These results suggest that suppressing the overexpression of Ca2+-handling and morphology-related molecules using isorhamnetin treatment may contribute to the prevention of AF vulnerability.