Oridonin Attenuates Diabetes‐Induced Renal Fibrosis via Inhibition of the TXNIP/NLRP3 and NF‐κB Pathway in Rats

Gengzhen Huang Chengdu University of Traditional Chinese Medicine Yaodan Zhang Hospital of Chengdu University of Traditional Chinese Medicine Yingying Zhang Chengdu University of Traditional Chinese Medicine Xiaotao Zhou Chengdu University of Traditional Chinese Medicine Yuan Xu Chengdu University of Traditional Chinese Medicine Huiting Wei Chengdu University of Traditional Chinese Medicine Yuerong Ma (  mayr666@163.com ) Chengdu University of Traditional Chinese Medicine


Introduction
Diabetes has become a global health concern in recent years. Diabetic nephropathy (DN) has been identi ed as a major cause of persistent kidney disease and end-stage renal failure [1,2] . The pathological symptoms of DN include the in ammatory in ltration of nephrocytes, damage of the renal tubular epithelial cells, glomerular sclerosis and apoptosis, and renal tubular interstitial brosis [3] .
Although DN has been considered primarily as a non-immune kidney disease in the past, recent theoretical and experimental studies have suggested that chronic in ammation and oxidative stress are two important contributors to the progression of DN [4,5] . The diabetic environment leads to the production of advanced glycation end products and changes in circulation and hemodynamics, followed by the release of reactive oxygen species (ROS) and in ammatory mediators, resulting in impaired renal function and increased markers of brosis [6] . Evidence collected in recent years suggests that in ammatory processes promoted by innate immune responses are critical in the pathogenesis of DN [7,8] .
TLR4 is a component of primary innate immune receptor-mediated in ammatory signaling pathway, which is distributed in glomerular mesangial cells and renal tubular epithelial cells in renal tissue [9,10] .
There is increasing evidence that TLR4 expression is signi cantly increased in renal tubular epithelial cells and mesangial cells in response to hyperglycemia and angiotensin II, which activates the downstream nuclear factor (NF -κB) pathway and accelerates secretion of pro-in ammatory cytokines.
Nod-like receptor protein-3 (NLRP3) in ammasome is an assembled molecular complex consisting of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and cysteinyl aspartate speci c proteinase (caspase-1) [13,14] . It plays an important role in the occurrence and development of various non-bacterial in ammatory diseases, especially those closely related to metabolic disorders and endogenous stimulation. In hyperglycemia, the body can produce a variety of dangerous related molecules, such as fatty acids and ROS, which can be recognized by the related pattern recognition receptor (PRR), thus initiating the activation of NLRP3 pathway. Upon the activation of in ammasomes, IL-1β and IL-18 are processed and activated, resulting in pro-in ammatory responses, which aggravate the in ammatory injury and decrease the kidney function. Studies have shown that direct or indirect inhibition of NLRP3 in ammasome activity is bene cial to reduce in ammatory injury and renal brosis [15] . NLRP3 in ammasome is a popular target in the treatment of diabetic nephropathy.
The Trx system is an important antioxidant system, which resizes oxidative stress by providing electrons to peroxides, thus enabling peroxides to effectively remove reactive oxygen species and nitrogen.
Thioredoxin-interacting protein (TXNIP) play an important role in cell death and immune response through interaction with Trx system. TXNIP is reported as a key regulator of pancreatic β-cell biological function [16] . The excess production of reactive oxygen species in diabetes leads to the separation of TXNIP from its binding protein Trx, which binds to NLRP3, leading to the activation of NLRP3 in ammasome [17] . It has been reported that NADPH oxidase (NOX4)-derived ROS promotes the dissociation of TXNIP-Trx and increases the binding of TXNIP to NLRP3 [18] . TXNIP might be the key to linking the hyperglycemic environment to in ammation by activating the NLRP3 in ammasome.
Oridonin (Ori, Figure 1) is a famous diterpenoid isolated from Rubescens rubescens, which has many biological properties such as antioxidant, anti-in ammatory, immunomodulatory and anti-tumor [19] . Ori has been used to treat in ammatory diseases in China for hundreds of years and has become one of the most popular herbs clinically. Evidence reported that Ori e ciently increases survival, alleviates proteinuria, attenuates renal disfunction and ameliorates the clinical manifestations of systemic lupus erythematosus (SLE) in mice [20] . Studies have also been reported the therapeutic effects of Ori in DN by reducing in ammatory cell in ltration, down-regulating TLR4 expression and inhibiting NF-κB and p38-MAPK activation with a type 2 diabetes mellitus (T2DM) rat model and HG-treated rat mesangial cells [21] .
However, few studies have investigated the effects of Ori on TXINP/NLRP3 in diabetic rats. Therefore, we aimed to investigate whether Ori protects against renal brosis in diabetic rats and further elucidate whether the anti-in ammatory mechanism that involves the TLR4/NF-κB and TXNIP/NLRP3 signaling pathways. Our ndings may support the clinical application of Ori as a treatment for DN.

Animals and treatment
Speci c pathogen-free grade male Sprague Dawley rats (weighing 150-200 g) were purchased from the Charles River Laboratory Animal Technology Co. Ltd. (Beijing, China). The rats were maintained under a standard temperature (24 °C), humidity (55 %) and 12-h light/dark cycle.
Four groups (8 rats per group) were included: a normal control (NC), T2DM model (DM), DM + Ori-10 (10mg/kg/day), and DM + Ori-20 (20mg/kg/day). The DM and DM + Ori groups were fed a high-fat diet (D12492) for 4 weeks to establish an insulin resistance model. All the insulin resistance model rats were converted into DM rats by a single intraperitoneal injection of STZ (35 mg/kg dissolved in 0.1 mol/l citric acid buffer, pH 4.3). At 72 h after STZ injection, we measured random blood glucose levels to con rm the successful establishment of the diabetes model. A random blood glucose level of >16.7 mmol/l after STZ injection was indicative of the establishment of the model. The rats in DM + Ori-10 and DM + Ori-20 groups were intraperitoneally injected with 10 or 20 mg/kg/day Ori, while the rats in the NC and DM groups received the equivalent dose of normal saline. The injection lasted for 2 weeks. All the rats were sacri ced under sodium pentobarbital anesthesia. Serum was then separated by centrifugation and stored at -80 ℃ for subsequent experiments. Kidney tissues (cortex) were also excised, weighed, and stored in liquid nitrogen or xed in 4% paraformaldehyde. The kidney weight-to-body weight ratio was calculated for each rat.

Assessment of biochemical markers
Scr, BUN and UA concentrations were determined using assay kits, according to the protocols provided by the manufacturers (Nanjing Jiancheng Institute of Biotechnology).

Histopathological examination of kidney tissues
Portions of the renal cortex xed in 4% paraformaldehyde were embedded in para n and cut into 5-μmthick sections. The tissue sections were stained with hematoxylin-eosin (H&E) and MASSON for assessment under a light microscope (Olympus, Japan).

Western blot analysis
Total protein was extracted from renal tissues, according to the manufacturer's protocol (Beyotime, China). Equal amounts of protein (50 μg) were separated by 8-10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred onto polyvinylidene uoride (PVDF) membranes, which were blocked with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 1 h and then incubated with primary antibodies for the following proteins overnight at 4 ℃. The membranes were then incubated with the appropriate secondary antibody, which was conjugated to horseradish peroxidase, for 1 h at room temperature. Protein bands were detected using an ECL Western Blotting Detection System (ImageQuant LAS, USA). GAPDH was used as a loading control.
Statistical analysis SPSS 22.0 software was used to analyze the data, which are presented as the mean ± SD. Differences between the groups were assessed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically signi cant.

Ori alleviated blood glucose and improved renal function in DM
An increased kidney weight/body weight ratio is a sign of swelling and damage to the kidneys. As shown in Fig. 2B, the kidney-to-body weight ratio in the DM group was signi cantly higher than that in the normal and Ori control groups (P < 0.05); Diabetic rats treated with Ori (10 and 20 mg/kg/day) for 4 weeks had a much lower kidney weight-to-body weight ratio than untreated diabetic rats (P < 0.05). Besides, there were signi cant differences in blood glucose levels between the DM and DM + Ori groups (P < 0.05, Fig. 2C).
Increases in the indicated renal functional parameters (Scr, BUN, UA) are considered hallmarks of the progression of renal disease. Scr, BUN, and UA concentrations in the DM group were higher than those in the normal and Ori control groups (Fig. 2D,E,F P < 0.05). Remarkably, those parameters were signi cantly decreased by treatment with Ori in the DM + Ori groups compared with those in the DM group, with a dose-dependent manner. These data showed that Ori protects the kidney in diabetic rats to some certain extent.

Ori attenuated renal histopathological injury in DM
The changes in renal histopathology in the different groups were shown in Fig. 3A. Sections from normal group showed normal renal structures. H&E and MASSON staining showed that the sections from the DM group displayed glomerular hypertrophy, mesangial matrix expansion, tubular dilation and interstitial in ammation in DM. However, Ori-10 and Ori-20 treatment signi cantly attenuated these diabetic histopathological alterations.

Ori inhibited renal brosis in DM
MASSON images (representative images in Fig. 3A) showed that the normal group had almost no blue staining, indicating that the area of brosis was very small, while the renal tissue of DM rats had renal tubulointerstitial and glomerular brosis. Masson's trichrome staining revealed signi cantly diminished brosis in Oridonin-treated rats than in saline-treated controls. The protein expression levels of TGF-β, α-SMA and Collagen-indicated that Oridonin (10 and 20 mg/kg/day) treatment reduced renal brosis in DM rats (Fig. 3B,C,D,E) (P < 0.05). These data showed that Ori protects the kidney from the progression of brosis in diabetic rats with a dose-dependent manner.

Ori inhibited TLR4/NF-κB activation in DM
TLR4/NF-κB is a key innate immune pathway involved in in ammatory processes. To assess the inhibitory effects of Ori on the in ammatory response, we examined the effects of Ori (10 and 20 mg/kg/day) on TLR4/NF-κB activity in renal tissues by western blot. As shown in Fig. 4A, TLR4 and NF-κB (p-p65) protein expression levels in the DM group were signi cantly higher than those in the normal and Ori-10 and Ori-20 groups (P < 0.05). The increase in NF-κB protein expression was attenuated by Ori treatment (Fig. 4B,C P < 0.05). These data showed that Ori protects the kidney from the activation of TLR4/NF-κB in diabetic rats with a dose-dependent manner.

Ori inhibited TXNIP/NLRP3 pathway in DM
To determine how Oridonin reduces renal brosis, we evaluated the expression of in ammatory factors known to promote brosis. TXNIP/NLRP3 has been shown to be involved in the progression of diabetic renal brosis. As previously reported, Ori is a covalent NLRP3 inhibitor [19] . As shown in Fig. 5A, TXNIP/NLRP3 pathway protein expression levels in the DM group were signi cantly higher than those in the normal (P < 0.05). Treatment with Ori (10 and 20 mg/kg/day) had reduced (P < 0.05) protein expression levels of TXNIP (Fig. 5B), NLRP3 (Fig. 5C), cleaved-caspase-1 (Fig. 5D), and cleaved-IL-1β (Fig.  5E). Given the mechanistic role of cleaved IL-1β in promoting brosis, these ndings also suggest that Ori reduces renal brosis by inhibiting NLRP3 and cleaved IL-1β release. These data showed that Ori protects the kidney from the activation of TXNIP/NLRP3 in diabetic rats with a dose-dependent manner for the rst time.

Discussion
Renal brosis is the key pathological change of diabetic nephropathy (DKD), and signi cantly increases the mortality of patients with advanced DKD. Different signaling pathways are involved in renal brosis, including the TGF-β, MAPK, PI3K/Akt, JAK/STAT, Wnt/β-catenin, and Notch pathways [3,4] . These pathways all play important roles in the accumulation of ECM, the expression of collagen and bronectin, and the secretion of other related proteins. In addition, more and more new therapies are being investigated in clinical trials, and many efforts have been made to delay or even attempt to reverse the progression of renal brosis [22][23][24] .
Evidence accumulated in recent years indicates that in ammation plays an important role in the occurrence and aggravation of DN kidney injury [7,8] . Increasing numbers of studies have shown that increases in in ammatory marker levels are related to the anti-DN effects of some renoprotective molecules [2] . However, the mechanisms behind these phenomena are not fully understood. Therefore, further study on the mechanism of renal immunity and in ammation and search for drugs to inhibit immune in ammatory response may nd new targets for DN anti-in ammatory therapy. Natural antiin ammatory products are a safe alternative to traditional methods for regulating in ammatory diseases [25][26][27][28] .
Li et al. showed that Ori exerts protective effect in diabetes-induced renal injury through the TLR4/NF-κB signaling pathways [21] . Lin et al. demonstrated that Ori reduced proteinuria and attenuated renal damage in a spontaneous SLE mouse model by regulating the in ammatory responses [20] . These results suggested that Ori attenuates proteinuria and protects the kidney from injury. Moreover, Ori also acts on a variety of cells, including immune cells, hepatocytes and vascular endothelial cells, to exert its protective effect. Bohanon et al. reported that Ori inhibited hepatic stellate cell proliferation and brogenesis by suppressing endogenous and TGF-β1-induced ECM proteins [29] . Current study demonstrated that oridonin inhibits collagen deposition and in ammation to attenuate CCl4-induced liver brosis in mice through inhibition of the NLRP3 in ammasome [30] . However, to date, whether Ori suppresses NLRP3 pathway and thus exerts bene cial effects on diabetes-induced renal brosis has not been explored. To con rm these effects, we investigated the inhibitory effects of Ori on the in ammatory response and brosis in a diabetic rat model. In our study, compared with rats in the control groups, rats in the DM group displayed increased plasma glucose levels, as well as an increased kidney weight-to-body weight ratio, which is indicative of renal injury. Signi cantly increased BUN, Scr, and UA concentrations were also noted in rats in the DM group. However, treatment with Ori effectively reversed these changes, as it lowered BUN, Scr, UA concentrations, and the kidney weight-to-body weight ratio. Previous studies have shown that Ori hardly affected plasma glucose levels. The discrepancy may be due to difference of the duration of the experiment. Previous experiment lasted for 12 weeks and we conducted for 6 weeks [21] .
The typical pathological changes of DN are mesangial cell proliferation, dilatation of renal tubules with accumulation of extracellular matrix and thickening of glomerular capillary wall, accompanied by nodular sclerosis, and eventually progressed to complete diabetic nephropathy. In this study, Ori treatment signi cantly ameliorated these diabetes-induced histopathological alterations. These data indicated that Ori improves renal function, ameliorates diabetes-induced renal injury and delays progressive nephrotoxicity in rats with DM.
The signi cance of the Trx-TXNIP signaling system is increasingly recognized [16] . Recent studies have shown a complex thiol-dependent interaction between TXNIP and the in ammation-related pathway of progressive diabetic nephropathy, the interaction of NLRP3 and TXNIP may be a signi cant signal of the formation of NOX4-derived NLRP3 in ammation in hyperhomocysteinemia-induced glomerular damage [31] . To verify whether the TXNIP-NLRP3 axis plays an important role in the therapeutic effects of Ori, we investigated the expression of NLRP3, TXNIP, and IL-1β. Our ndings con rm the previous view that hyperglycemia-induced mitochondrial dysfunction plays an important role in the occurrence of DN, and also con rm to some extent that Ori can regulate TXNIP-NLRP3 axis to affect ROS levels to ameliorate mitochondrial damage.
In DN, the NLRP3 in ammasome is an intracellular platform that converts pro-IL-1β into active forms (IL-1β p17) responding to danger signals and triggers in ammatory programmed cell death. In recent studies, MCC950 (inhibitor of NLRP3) reduced liver in ammation and brosis by suppression collagen I, α-SMA and hepatic connective tissue growth factor expression in a mouse model of non-alcoholic steatohepatitis [32] . TGF-β1 is a major cytokine secreted by mesangial cells that mediates the development of DN [33] . TGF-β1 is a key cytokine mediating collagen deposition in kidney, including promoting the production of ECM, inhibiting the degradation of ECM and participating in renal brosis [34,35] . ECM of DN patients is produced by mesangial cells and mainly consists of bronectin, type IV collagen and a small amount of type I collagen. α-SMA was weakly expressed in normal mesangial cells, but was signi cantly increased under high glucose stimulation. IL-1β has been demonstrated to stimulate production of TGF-β1, bronectin, collagen I and mesangial proliferation. Our experimental results showed that Ori has a good inhibitory effect on these indicators promoting renal brosis.
In conclusion, our present study suggests that oridonin inhibits collagen deposition and in ammation, thereby alleviating diabetic induced renal injury and brosis in rats. However, the experiments conducted in this study covered only a narrow and super cial scope of pharmacological identi cation. Therefore, the renal protective effect of Ori remains to be further studied. These results should also be compared with current rst-line drugs for renal brosis.

Declarations Ethics approval and consent to participate
Animal experiments were carried out in accordance with the National Animal Protection and Use Guidelines and approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine.

Consent for publication
Authors are responsible for correctness of the statements provided in the manuscript.
Availability of data and materials Figures Figure 1 The chemical structure of Oridonin