Mechanism of crocin regulating NLRP3 pathway and inhibiting epithelial mesenchymal transition in the treatment of diabetes nephropathy
Diabetes kidney disease (DKD) is a common complication of diabetes, which will lead to damage to the structure and function of the kidney, mainly manifested as glomerular mesangial dilatation, tubulointerstitial fibrosis, glomerular basement membrane thickening, etc., and eventually develop into end-stage kidney disease. DKD has a hidden onset, rapid progression, complex mechanism, and poor prognosis. Western medicine treatment mainly focuses on controlling blood sugar, blood pressure, and blood lipids, but the progression of DKD does not slow down, seriously affecting the quality of life of patients. Moreover, as the incidence rate of DKD increases year by year in China, DKD has brought huge economic and social burdens, and effective drugs for treating DKD need to be developed urgently.
The active ingredients of traditional Chinese medicine have significant advantages in delaying the progression of DKD. Epimedium glycoside can alleviate inflammation in renal tissue and delay the progression of diabetic kidney disease (DKD) by inhibiting the nuclear factor kappaB (NF – κ B) pathway. Silymarin can activate the protein kinase B (Akt) pathway to improve DKD. Hypericin can improve mouse DKD by promoting polarization of macrophages from M1 to M2 phenotype and differentiation of CD4+T cells into helper T cell 2 (Th2) and regulatory T cell (Treg) populations. The bioactive component of licorice, licorice flavonoids, can improve diabetic kidney disease (DKD) by inhibiting iron death and the vascular endothelial growth factor (VEGF)/Akt/extracellular signal regulated kinase (ERK) pathway. Paeoniflorin can inhibit various inflammatory factors such as interleukin-1 beta (IL-1 β), downregulate the protein expression of phosphorylated Janus kinase 2 (p-JAK2) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in renal cells, alleviate renal tissue inflammation and autophagy response, and protect the kidneys.
Crocin (CRO) is the main active ingredient of traditional Chinese medicine saffron, which has various effects such as anti-inflammatory and antioxidant. Studies have found that CRO can alleviate DKD by inhibiting oxidative stress and inflammatory response. There are also studies that have found that CRO can inhibit the activation of the NLRP3 pathway in cardiomyocytes. However, it is unclear whether it can treat DKD by inhibiting the activation of NOD like receptor thermal protein domain associated protein 3 (NLRP3) and suppressing epithelial mesenchymal transition (EMT). Therefore, this study established a DKD mouse model to investigate the therapeutic effect of CRO on DKD mice, and studied the mechanism of CRO in treating DKD from the perspectives of EMT and NLRP3 pathways.

DKD is a microvascular disease caused by diabetes, which leads to glomerulosclerosis, thickening of basement membrane, and fibrosis of renal interstitium. It is an important cause of end-stage renal disease. The increasing incidence rate year by year has become a serious medical burden. Using animal models to study DKD is conducive to the development of new drugs, thus improving the quality of life of patients and reducing the social burden. The combination of HFD diet and STZ injection induction is a classic method for modeling DKD, and after successful modeling, the symptoms are similar to those of DKD patients. The principle is mainly related to the induction of insulin resistance in mice by HFD diet and the induction of pancreatic beta cell necrosis and kidney damage by STZ injection. 24-hour UTP, Cr, and BUN are commonly used to evaluate glomerular filtration function, and elevated levels indicate renal dysfunction. In this study, DKD mice showed severe renal lesions, including increased glomeruli, inflammatory infiltration of renal tubulointerstitial tissue accompanied by fibrosis, indicating successful modeling. After CRO intervention, the above symptoms were improved, with high-dose CRO showing the most significant improvement effect, and the degree of improvement was similar to that of the positive drug MCC950. Other studies have shown that CRO intervention at a dose of 25mg/kg can effectively improve bleomycin induced pulmonary fibrosis.
EMT is the direct cause of renal fibrosis and proteinuria, and plays an important role in the development of DKD. Studies have shown that CRO can inhibit EMT in the liver to improve liver fibrosis. Therefore, this study further explores whether CRO can improve renal EMT. In this study, the expression of E-cad in the kidneys of DKD mice was downregulated, while the expression of VIM, α – SMA, and TGF – β 1 was upregulated, indicating an exacerbation of EMT in the kidneys of DKD mice. E-cad is the main marker of epithelial cells, which plays a role in maintaining epithelial cell polarity and intercellular adhesion. Interstitial cells mainly express VIM, α – SMA, and TGF – β 1, among which TGF – β 1 has a strong pro fibrotic effect. Numerous studies have confirmed that TGF – β 1 is highly expressed in DKD kidney tissue. During the EMT process, the decrease or disappearance of epithelial cell adhesion molecules and cytoskeletal components leads to a phenotypic transformation of epithelial cells, obtaining markers of stromal cells. Under various injury stimuli, differentiated epithelial cells undergo phenotypic transformation to form a large number of fibroblasts and myofibroblasts, promoting the development of renal fibrosis. The results of this study found that CRO can upregulate the expression of E-cad, downregulate the expression of VIM, α – SMA, and TGF – β 1, and play a good inhibitory role in the development of renal EM. In addition, CRO has been shown to inhibit EMT in tumor cells through the MAPK/ERK pathway. MAPK/ERK is also an important factor leading to renal fibrosis in diabetic kidney disease. In the future, further exploration can be conducted on the inhibitory effect of CRO on MAPK/ERK induced renal fibrosis, in order to find new targets for CRO to inhibit EMT.
The lesions of EMT are dynamic and reversible, and studies have shown that the NLRP3 pathway is involved in regulating its phenotype transition. In this study, NLRP3 inflammasome was highly expressed in the kidneys of DKD mice, exacerbating the inflammatory response in the kidneys of DKD mice. The NLRP3 inflammasome is a multi protein complex composed of NLRP3, ASC, and caspase-1. Activated NLRP3 inflammasome plays an important role in the upstream of DKD. When NLRP3 is activated, it promotes the assembly of NLRP3 inflammasome components (NLRP3, ASC, caspase-1), thereby activating pro Caspase-1 to form active cleaned-Caspase-1, which cleaves and matures inflammatory factors IL-1 β and IL-18. Mature-IL-1 β can stimulate the synthesis of TGF – β 1, which is a key factor in the development of EMT. TGF – β 1 can promote the synthesis of various protein components in the extracellular matrix and prevent their degradation, thereby exacerbating the process of renal EMT and accelerating the formation of renal fibrosis. In addition, studies have shown that the expression of α – SMA in NLRP3 knockout mice is significantly downregulated. Overexpression of NLRP3 inhibits the expression of E-cad and promotes the expression of α – SMA, while VIM has been shown to promote the activation of NLRP3. All of these suggest that there is a mutual regulatory effect between NLRP3 and EMT, and inhibiting NLRP3 can significantly alleviate renal EMT. And this study found that CRO intervention effectively inhibits the expression of NLRP3 inflammasome related factors, exerting good anti-inflammatory effects, while also exerting anti EMT effects. The activation of NLRP3 requires the involvement of dual signals. Firstly, the transcription factor NF – κ B is activated and enters the nucleus, promoting the expression of genes such as NLRP3 and IL-1 β (first signal); Afterwards, under the stimulation of factors such as K+and ROS, NLRP3 inflammasome assembly (second signal) is initiated. Interestingly, previous studies have shown the inhibitory effect of CRO on NF – κ B. So whether CRO can suppress NLRP3 induced EMT and improve DKD by inhibiting NF – κ B (the first signal) can become a future research direction. CRO can inhibit NLRP3 activation and improve peritonitis by reducing mitochondrial reactive oxygen species production. Meanwhile, the antioxidant and mitochondrial repair functions of CRO have been reported in various models. So, further research is needed to confirm whether CRO can improve the EMT of DKD model by reducing mitochondrial ROS production, thereby blocking the activation of the second signal of NLRP3 and inhibiting its activation. In addition, this study selected the NLRP3 inhibitor MCC950 as a positive control and found that CRO exerted anti-inflammatory effects similar to MCC950. In summary, CRO can ultimately alleviate DKD by inhibiting the NLRP3 pathway to suppress EMT.