Curcumin is a dione-type compound produced by the rhizomes of turmeric, and it has multiple well-known bioactivities, such as antioxidative, antifibrogenic, anti-inflammatory and anti-apoptosis activities. Many scientists have reported the oral therapeutic effects of curcumin, such as hepatic, pulmonary and renal fibrosis [18–20]. Sericin is a novel natural product protein extracted from silkworm cocoon, and it has good anti-inflammatory performance and biocompatibility. Oral sericin has good anti-inflammatory bioactivity and cell repair effects [21–22]. To inhibit EMT-related pathways and suppress the progression of renal interstitial fibrosis, we constructed sericin-curcumin nanoconjugates that could be taken orally.
From the morphology of the nanoconjugates, it can be concluded that GA crosslinked sericin and curcumin tightly into an aggregated state at the center part, and scattered parts were crosslinked relatively weakly (Fig. 2A). The successful assembly of sericin and curcumin was further verified by Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance spectroscopy (1H NMR) and X-ray diffraction (XRD) (Fig. 2B-D, Figure S1). Based on the FTIR patterns, we concluded that the nanoconjugates had been successfully synthesized. 1H NMR also confirmed the successful connection of sericin with curcumin.
Sericin accounts for 20–30% of the total weight of silk. When the silkworm prepares its cocoon, sericin plays the role of adhesion, wrapping two monofilaments together to form the cocoon silk [23]. Sericin contains a large number of amino acids whose side chains have hydrophilic groups, such as serine, aspartic acid, etc., conferring good water solubility [24], but we found that sericin is hardly soluble in ethanol, and sericin quickly precipitates in a suspension of ethanol. The maximum absorption peak for sericin in both water and ethanol was at 275 nm (Fig. 3C, F). Curcumin is insoluble in water, and it quickly floats water suspensions. The maximum absorption peaks of curcumin in ethanol were at 264 and 426 nm, but curcumin exhibited a broad absorption band from 300 to 550 nm in water (Fig. 3B, E). The nanoconjugates can be dissolved in ethanol and water. It can be observed that the UV absorption curve of nanoconjugates dissolved in ethanol became steeper and straighter at 264–288 nm than that of curcumin because of the addition of sericin. More importantly, the maximum absorption peaks of nanoconjugates dissolved in water were observed at 275 and 405 nm, and the absorption band at approximately 405 nm was broader than the absorption band of curcumin dissolved in ethanol (Fig. 3A, D). These results illustrated that we successfully crosslinked sericin and curcumin.
TGF-β1 is the core cytokine of renal interstitial fibrosis, and its expression can be stimulated by the internal environmental state of CKD. Cell photographs and histopathological patterns proved that the sericin-curcumin nanoconjugate had good anti-fibrosis performance (Fig. 4b, 5b). To verify the biomedical antifibrotic mechanism of the nanoconjugates, we measured the gene expression levels of TGF-β1 and snail-1. Snail-1 is the downstream molecule of the TGF-β1/Smad pathways. The gene expression levels of TGF-β1 and snail-1 in TGF-β1-stimulated HK-2 cells and UUO mouse renal cortex were elevated, nanoconjugates evidently reversed this effect, and curcumin and sericin also reversed the changes in the gene expression of TGF-β1 and snail-1 to a certain degree both in vivo and in vitro.
Curcumin can enter cells to inhibit the phosphorylation of Smad 1/2 [25], and we speculated that the flavonoid-rich ethanolic from sericin in nanoconjugates could also exert its anti-fibrosis effect by inhibiting the phosphorylation of Smad 1/2. Curcumin and sericin may have synergistic antifibrotic effects. Western blotting assays were used to verify whether sericin can repress the phosphorylation of Smad 1/2. The results showed that both TGF-β1 and p-Smad1/2 were notably decreased in the renal cortex and cell homogenates in nanoconjugate-treated groups, and curcumin and sericin also reversed these effects on the protein expression levels of TGF-β1 and p-Smad1/2 to a certain degree both in vivo and in vitro. We concluded that flavonoid-rich ethanol from sericin also played an antifibrotic role by suppressing the phosphorylation of Smad 1/2, and then, the fibrogenic effect of TGF-β1 was eliminated by reducing the levels of phosphorylated Smad1/2. The degree of nanoconjugate digestion in animals and the mechanism of nanoconjugate degradation need to be further studied. In addition, we observed that there were significant differences in animal activity levels among the different groups. The animal activity levels of the UUO + NANO and UUO + SER groups were equivalent to those of the negative control group, but this performance was poor in the UUO group and UUO + CUR group (data not shown);perhaps the mice in the UUO group and UUO + CUR group suffered from depression [26].