Diabetes nephropathy (DN) is the most common microvascular complication of diabetes and the most common cause of end-stage renal disease (ESRD). The current pathogenesis of diabetic nephropathy is complex, involving changes in renal hemodynamics, ischemia, oxidative stress, inflammatory activation, and activation of the renin angiotensin aldosterone system (RAAS), leading to renal fibrosis and ultimately affecting renal function. Timely blocking renal fibrosis can effectively prevent DN from progressing to ESRD.

The overactivation of the RhoA/ROCK signaling pathway mediates fibrosis processes in various diseases, including diabetic nephropathy, and ROCK inhibitors represented by fasudil have shown good anti DN renal fibrosis effects. Naringenin (NRG) is a kind of flavonoid compound purified from citrus plants, which has a variety of effects, such as anti arrhythmia, anti atherosclerosis, anti-inflammatory, anti fibrosis, anti-virus, anti-tumor and so on. It is widely used in in vivo and in vitro research. However, there is currently limited research on the role of naringin in diabetic nephropathy (DN). Therefore, in this study, a DN mouse model was replicated and treated with naringin to observe the progression of DN renal fibrosis. At the same time, we set up fasudil as a positive control group to further explore the effect of naringin on the RhoA/ROCK signaling pathway.

32 C57BL/6 mice were randomly divided into a normal group, a model group, a naringin group, and a fasudil group. After successful replication of the DN model, the mice were treated with naringin and fasudil, while the normal group and model group were treated with physiological saline control. The mice were euthanized for 12 consecutive weeks to observe changes in serum creatinine (Scr), 24-hour urinary protein quantification (24-hour Upro), blood glucose, and body weight; HE and Masson staining were used to observe the morphological changes in the kidneys; Immunohistochemistry was used to observe the levels of RhoA, ROCK1, ROCK2, Type-I collagen (Col1), and Type-III collagen (Col3) in the kidneys; Western blot analysis of renal RhoA ROCK1、ROCK2、p-MYPT1、 The levels of laminin (LN) and fibronectin (FN)

The early pathological changes of DN nephropathy are mainly glomerulosclerosis, renal arteriole damage, and tubular degeneration, gradually leading to renal fibrosis. In the late stage, there is a large amount of proteinuria and renal failure. Naringin is one of the representative drugs of flavonoids. Wenli et al. found that naringin may alleviate renal fibrosis and protect renal function in DN rats by downregulating the TGF – β 1/smad signaling pathway. Further research by Ning et al. found that naringin upregulates the expression of let-7a, which negatively regulates transforming growth factor – β 1 receptor 1 (TGFBR1), thereby inhibiting the TGF – β 1/smad signaling pathway and treating diabetic nephropathy. In addition, naringin may also slow down the progression of diabetic nephropathy and protect kidney function by downregulating oxidative stress levels, inhibiting the expression of inflammatory factors, and promoting cell apoptosis. In this study, type 1 diabetes mouse model was established by intraperitoneal injection of STZ, and then continued to feed for 4 weeks, and 24 h Upro was twice as large as before modeling, which was regarded as successful replication of DN model. After modeling, naringenin 50 mg/(kg · d FN expression was reduced, renal fibrosis was reduced, proteinuria and renal function were improved, and blood sugar and body weight had no significant change compared with the model group, indicating that naringenin and fasudil could improve DN renal fibrosis without affecting body weight and blood sugar.

RhoA is a member of the Ras superfamily in small molecule guanosine triphosphate (GTP) binding proteins. Tyrosine kinases and G protein coupled receptors can recruit and activate guanine nucleotide exchange factors (GEF) of RhoA, thereby converting RhoA from the inactive state of GDP binding to the active state of GTP binding. RhoA plays the role of a molecular switch to achieve the transition between the two states, acting on downstream signaling molecules to produce biological effects. Rho associated protein kinase (ROCK) is the first downstream effector molecule of RhoA discovered and currently the most functionally studied. The relative molecular weight of ROCK is approximately 160 kDa, belonging to the serine/threonine protein kinase family system. There are two subtypes of ROCK: ROCK1 and ROCK2, both of which have three main structural domains: a catalytic kinase domain at the N-terminus, a coiled helix domain containing a Rho binding domain (RBD) in the middle, and a pH domain rich in cysteine (CRD) at the C-terminus. The 65% amino acid sequences of ROCK1 and ROCK2 are identical, and the homology of kinase domains can reach 92%. ROCK is expressed in most tissues, but there is heterogeneity among different tissues. ROCK1 is mainly expressed in the liver, testes, spleen, kidneys, lungs, etc., while ROCK2 is mainly expressed in the brain, skeletal muscle, heart, etc. The signal pathway composed of RhoA/ROCK participates in the regulation of physiological activities such as cell proliferation, adhesion, migration, and apoptosis through interactions with downstream substrates such as myosin light chain (MLC), MYPT-1, CPI-17, ERM, etc. In pathological conditions, the RhoA/ROCK signaling pathway upregulates the secretion of various pro fibrotic factors, such as platelet-derived growth factor (PDGF), angiotensin II (AngII), endothelin-1 (ET-1), monocyte chemoattractant factor (MCP-1), interleukin-6 (IL-6), transforming growth factor – β 1 (TGF – β 1), connective tissue growth factor (CTGF), etc. ROCK can also induce the activation of nuclear factor kappa B (NF – κ B), which in turn upregulates the secretion of pro fibrotic factors such as tumor necrosis factor (TNF – α) and IL-1 β. Fasudil is currently the first and only approved ROCK inhibitor for clinical use. Due to its powerful vasodilatory effect, it is widely used in vascular spasm diseases such as subarachnoid hemorrhage and ischemic heart disease. It can effectively bind to ATP dependent kinase domains to block ROCK and inhibit the signal transduction of RhoA/ROCK signaling pathway. In the field of diabetic nephropathy, fasudil has also been widely studied and improves DN by affecting multiple pathways, including: (1) inhibiting the activation of NF – κ B, FN, and TGF – β, reducing inflammatory response, and alleviating glomerulosclerosis; (2) Promote the formation of podocyte cells, restore abnormal contraction movement, reduce glomerular filtration membrane permeability and proteinuria formation; (3) Inhibit the epithelial mesenchymal transition (EMT) process of renal tubular epithelial cells and weaken renal interstitial fibrosis; (4) Expand the renal vascular network, improve renal hemodynamics, and reduce renal damage. In this study, compared with the normal group, the expression of RhoA, ROCK1, and ROCK2 in the kidney tissue of the model group mice significantly increased, the phosphorylation level of MYPT1 Thr853 increased, and the activity of ROCK increased; Compared with the model group, the expression of RhoA, ROCK1, and ROCK2 in renal tissue was significantly decreased in the Fasudil group, the phosphorylation level of MYPT1 Thr853 was reduced, the activity of ROCK was weakened, and there was a decrease in urinary protein and improvement in renal function; Similar to the fasudil group, the naringin group showed a significant decrease in the expression of RhoA, ROCK1, and ROCK2 in renal tissue compared to the model group. The phosphorylation level of MYPT1 Thr853 was reduced, and the activity of ROCK was weakened, accompanied by a decrease in urinary protein and improvement in renal function. However, there was no statistically significant difference in various indicators between the fasudil group and the naringin group. This experimental result suggests that the RhoA/ROCK1 signaling pathway is involved in the formation of renal fibrosis in DN mice, and naringin’s improvement of renal fibrosis in DN mice may be related to the downregulation of the RhoA/ROCK1 signaling pathway.

In summary, animal experimental studies suggest that naringin can significantly reduce the degree of renal fibrosis in DN mice, and its mechanism may be related to downregulation of the RhoA/ROCK signaling pathway. However, its specific mechanism and site of action require further in-depth research at the cellular and molecular levels.