Astragalosides IV Improves Palmitic Acid-induced Renal Tubular Epithelial Cells Injury Due to Inhibition of NLRP3 Inammasome

Diabetic nephropathy (DN) is a serious complication of type 2 diabetes mellitus (T2DM). Hyperlipidemia plays a key role in occurrence and development of DN. The main saturated fatty acids of palmitic acid (PA) is closely related to glomeruli and tubules injury in T2DM. Astragaloside IV (AS-IV) has comprehensive pharmacological effects, such as anti-inammation, anti-oxidation and anti-apoptosis. However, whether AS-IV can attenuate PA-induced renal tubular epithelial cells damages and its underlying mechanisms remain unclear.


Background
Diabetic nephropathy (DN) is a lethal complication of diabetes mellitus and has become a major cause of end-stage renal disease. Although numerous studies have focused the lesions of DN mainly on glomerulosclerosis, more and more studies indicated that diabetic renal damage also occurs in the renal tubules [1,2]. Increasing evidences demonstrate that renal tubular epithelial cells injury is also an important characteristic of DN, and apoptosis of renal tubular epithelial cells is observed in mice, rats and humans with DN [3]. In addition to hyperglycemia, high levels of free fatty acids (FFAs) are also observed in type 2 diabetes mellitus (T2DM) and have been identi ed as a risk factor of DN [4]. Recent studies indicated that high levels of FFAs are involved in DN by inducing endoplasmic reticulum stress and oxidative stress, and leading to podocyte apoptosis [5]. Clinical studies have found that many patients with DN have obvious dyslipidemia [6,7]. Palmitic acid (PA) is a major lipid derivative in human body, and it has been reported that the lipotoxicity induced by PA can cause human kidney-2 (HK-2) epithelial cell to produce oxidative stress, in ammation, brosis and apoptosis, thereby inducing renal dysfunction and aggravating DN [8]. However, the speci c mechanism of PA-induced renal tubular damage has not yet been elucidated.
Oxidative stress and in ammation play important roles in the pathological damage of renal tubules.
Excessive ROS production can induce oxidative stress and in ammation, and promote the renal tubular cells damage, resulting in renal injury [9]. NADPH oxidase (NOX) is a constitutively multi-subunit enzyme that acts as an oxygen sensor and an electron donor to generate ROS from molecular oxygen. NOX4, one subtype of NOX in kidney, contributes to the ROS accumulation in kidney diseases including DN [10,11].
Increasing researches have indicated that NOX4 is involved in the damage of HK-2 cells, leading to mesenchymal transition, apoptosis and in ammation [12,13]. Moreover, it has been reported that DN may be an in ammatory disease [14]. In ammasomes play a central role in cleaving procaspase-1 to caspase-1, which eventually leading to the generation of proin ammatory factors [15]. The subtype of NLRP3 in ammasome is widely expressed in the glomerulus and renal tubules [16]. It has been reported that NLRP3 in ammasome plays an important role in renal injury and closely involves in the pathogenesis of DN, acute and chronic kidney disease [17,18]. Over activation of NLRP3 in ammasome participates in the damage of renal tubular epithelial cells by releasing mature in ammatory factors [19].
ROS is one of the important factors for activation of in ammasome, and more and more evidence shows that NOX4 and NLRP3 in ammasome can regulate each other and play a role together in various kidney injuries [20,21]. Our previous works showed that PA exposure could signi cantly induce ROS accumulation in HK-2 cells [22]. Therefore, we hypothesized that NOX4-mediated activation of NLRP3 in ammasome may be involved in PA-induced HK-2 cells injury.
At present, there are few medicines for treatment of DN. Astragaloside IV (AS-IV) is extracted from traditional Chinese medicinal plant Astragalus membranaceus. In recent years, various pharmacological effects of AS-IV have been reported, such as anti-oxidative stress, anti-in ammatory, anti-apoptosis and anti-brosis in vivo and in vitro [23]. Growing evidence has shown that AS-IV can improve DN rats through antioxidant and anti-in ammatory mechanisms. Additionally, AS-IV can also inhibit high glucose-induced HK-2 cells apoptosis through inhibiting oxidative stress [1]. Our previous work indicated that AS-IV could also alleviate PA-induced HK-2 cells injury [22]. However, whether AS-IV can protect against kidney tubule injury by inhibition of NOX4-mediated NLRP3 in ammasome activation is still unknown. This study was performed to study the potential protective effect and mechanism of AS-IV on PA-induced HK-2 cells injury and on kidney tubular damage in T2DM rats. This research may provide potential targets for the prevention of DN.

Cell culture and treatment
Human kidney 2 (HK-2) is a proximal tubular cell line derived from normal kidney. HK-2 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100units/ml penicillin and 100 µg/ml streptomycin at 37 °C with 5% CO 2 . The experiments were divided into two parts. In the rst part, the HK-2 cells were treated with PA (200 µM) for 6, 9, 12, and 24 h respectively to study the effect and mechanism of PA on tubular cells injury. In the second part, to study the protective effect and mechanism of AS-IV on PA-induced tubular cells injury, the HK-2 cells were divided into six groups: (1) control group, the cells were cultured with 0.2% BSA; (2) model group, the cells were treated with PA (200 µM) for 24 h; (3-5) AS-IV groups, the cells were treated simultaneously with PA (200 µM) and AS-IV (10, 20 or 40 µM) for 24 h; (6) apocynin (Apo) group, the cells were treated simultaneously with PA (200 µM) and Apo (100 µM) for 24 h.
Palmitate (PA, sigam-aldrich, USA) was prepared as previously reported method [22]. Brie y, PA (27 mM) was dissolved in distilled water (70℃) for 30 min. The bovine serum albumin (BSA, 30%; Shanghai Yeasen Biotechnology Co., Ltd, China) was also dissolved in distilled water (55℃) for 30 min. Then the PA and BSA solution were mixed at a molar ratio of 6:1 and stored at 4℃ as a storage solution.
Astragaloside IV (AS-IV, ≥ 98%, Nanjing Zelang Pharmaceutical Technology Co.) and apocynin (Apo, Merck Millipore) were dissolved in DMSO as a stock solution stored at -20℃. When using, the stock solution was diluted with DMEM with nal DMSO concentration less 0.5% (v/v).
Eight weeks later, the HFD rats were administrated with a single intraperitoneal injection of streptozotocin (STZ, 35 mg/kg, Shanghai Yuanye Biotechnology Co., Ltd. Shanghai, China), which was dissolved in the citrate buffer (pH 4.6, 0.1 M). After 72 hours, the blood glucose concentrations were detected with a Roche blood glucose meter from the tail vein. The rats (blood glucose concentration ≥ 16.7 mmol/L) were divided randomly into three groups (n = 10): model group, AS-IV group (80 mg/kg) and Metformin (Met, 200 mg/kg) groups, and continued to be fed with HFD. AS-IV and Metformin (Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.) were suspended in carboxymethyl cellulose sodium (0.5%) and were treated intragastrically once a day. The normal control group and the model group were treated with equal volume of solvent. All animal experiments were approved by the Institutional Ethics Committee of Anhui Medical University.

Cell viability assay
Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Kumamoto, Japan) was used to assess cell viability. The cells (5 × 10 3 per well) were seeded in 96-well plate. The CCK-8 solution (10%) was added to the media and continuously incubated for 2 h. The optical density (OD, 450 nm) of each well was detected using a microplate reader (Thermo Fisher Company, USA). Cell viability = (administration group OD -blank group OD) / (control group OD -blank group OD) × 100%.

Oil Red O (ORO) staining of HK-2 cells
The cellular lipid deposition was detected by ORO Staining assay kit (Solarbio, Beijing). The cells were washed twice with PBS and then xed with ORO Fixative for 30 min. Then cells were washed twice with water and immersed in 60% isopropyl alcohol for 5 min. Afterwards, the cells were immersed in ORO Stain for 20 min and washed with water, and visualized by using a microscope (BX-51, Olympus Co., Ltd. Japan). The mean density of lipid deposition in each group were quanti ed by using Image-Pro Plus 6.0 software.

Apoptosis analysis of HK-2 cells
Annexin V-FITC/PI staining was used to detect the cell apoptosis by a commercial kits (Beibo Biology Co., Ltd. Shanghai, China). Brie y, the cells were suspended with 400 µL of 1 × binding buffer to make the cell concentration approximately 1 × 10 6 cells/ml. Then the cells were incubated with Annexin V-FITC and PI for 15 minutes in darkness at 2-8℃. Flow cytometry (BD Biosciences, USA) was used for analysis, and apoptotic HK-2 cells (Annexin V + /PI − ) were shown in the lower right quadrant.

ROS production assay
For ROS assay, HK-2 cells were incubated with H2DCFDA (KeyGEN, Jiangsu, China, 5 µM) for 30 min at 37℃ in darkness. The non uorescent H2DCFDA can be oxidized into DCF to generate uorescence by the existing peroxides, hydroperoxides, etc. after entering the cell. The results were examined by using a uorescence microscopy (Olympus IX71, Japan). The mean uorescence density of each group was analyzed by using the Image Pro Plus 6.0 software to assess the production of ROS.

Immuno uorescence staining
The cells were cultured in 24-well plates. For examination, the cells were washed three times with PBS, and then xed with paraformaldehyde (4%) for 30 min. After that, cells were permeabilized with 10% Triton X-100 for 30 min and blocked with 3% BSA for 30 min. Primary antibody against NLRP3 (1:200) was incubated overnight at 4℃, followed by incubation with a uorescent secondary antibody. Then cells were washed by PBS and stained nucleus with DAPI for 5 min. The positive areas were examined by using a microscopy (Olympus IX71, Japan). The mean uorescence density of each group was performed by using the Image Pro Plus 6.0 software to indicate the expression of NLRP3.

Enzyme-linked immunosorbent assay (ELISA)
The ELISA kits (MEIMIAN, Jiangsu, China) were conducted to assess the levels of interleukin IL-1β and IL-18 in cultured supernatants, and GSH, SOD in cells protein according to the protocols. All human ELISA kits had high sensitivity and excellent speci city for detection of IL-1β, IL-18, GSH and SOD with no signi cant cross-reactivity or interference being observed.

HE and Oil Red O staining of kidney tissues
The T2DM rats were sacri ced and the kidneys were removed immediately. One kidney was kept in -80 ℃, and the other was xed with paraformaldehyde (4%). For HE staining, the kidney specimen was embedded in para n, and cut into 5 µm thick sections. The para n sections conventional dewaxing to water and stain with hematoxylin and eosin (HE). For Oil Red O staining, kidney specimen was embedded in the OTC and cut into 10 µm sections with a frozen slicer (Leica CM3050, Germany). Kidney sections were stained with Oil Red O solution for 8-10 min, and observed under an optical microscope (Olympus IX71, Japan). The density of positive area in each group was analyzed with Image-Pro Plus 6.0 analysis software to quantify the lipid deposition.

Immunohistochemistry
Kidney sections were depara nized and rehydrated, then were placed in sodium citrate buffer (pH6.0) with a microwave oven heating for 10 min to repair antigen. Then the slides were incubated with 3% hydrogen peroxide solution for 25 min, then were blocked with 3% BSA for 30 min. Primary antibodies of NLRP3 (A nity, DF7438, 1:50) and NOX4 (Service bio, GB11347, 1:100) were added to the sections, and the sections were placed in a wet box and incubated at 4℃ overnight. Then the tissues were covered with secondary antibody (A nity, S0001, 1:500) and incubated at room temperature for 50 min. Subsequently, DAB chromogen (Service bio, G1211) was used and the nucleus was counter stained with hematoxylin.
The intensity of positive areas was analyzed by using Image-Pro Plus 6.0 analysis software to assess the expressions of NLRP3 and NOX4 in kidney tubules.

Statistical analysis
The data were analyzed using Graph Pad Prism 6 (Graph Pad Software, San Diego, CA, USA). The results were expressed as mean ± SD. Differences among groups were calculated using one-way analysis of variance (ANOVA), then followed by t-test to compare the differences between groups. P < 0.05 is de ned as statistically signi cant.

AS-IV attenuates lipid deposition in PA-induced HK-2 cells
Excessive lipid deposition in cells can cause lipotoxicity. To evaluate the effects of PA and AS-IV on cell lipid deposition, the Oil Red O Staining was performed in PA-induced HK-2 cells. As shown in Fig. 2A and B, the intracellular lipid deposition was signi cantly increased in HK-2 cells with the prolongation of PA exposure as compared with BSA control group (P < 0.01). As shown in Fig. 2C and D, AS-IV (10, 20 and 40 µM) and Apo treatment for 24 h signi cantly decreased lipid deposition in PA-induced HK-2 cells as compared with the model group (P < 0.01). The data suggested that AS-IV treatment could signi cantly alleviate PA-induced lipotoxicity in HK-2 cells.

AS-IV reduces apoptosis in PA-induced HK-2 cells
The renal tubular epithelial cells apoptosis also plays important roles in the pathogenesis of DN. To observe the effect of AS-IV on PA-induced HK-2 cells apoptosis, the cells were subjected to apoptosis assay by using Annexin V-FITC/PI Kits and were detected by ow cytometry. The results showed that the apoptosis rate was signi cantly increased with prolongation of PA exposure compared with BSA control group, especially in 12 h and 24 h (Fig. 3A and B, P < 0.05). However, as shown in Fig. 3C and D, AS-IV (20 and 40 µM) and Apo treatment signi cantly decreased the apoptosis rate in PA-induced HK-2 cells (P < 0.01). The results suggested that AS-IV treatment could inhibit PA-induced HK-2 cells apoptosis.

AS-IV decreases ROS generation in PA-induced HK-2 cells
The lipotoxicity caused by excessive lipid deposition can induce oxidative stress in the body. Therefore, we further performed H2DCFDA staining to evaluate PA-induced ROS accumulation in HK-2 cells. As shown in Fig. 4A and B, the results showed that the ROS level was signi cantly increased with increase of PA exposure time compared with BSA control group (P < 0.05 or P < 0.01). We also observed the effect of AS-IV and Apo treatment on PA-induced ROS accumulation in HK-2 cells. The results showed that AS-IV (10, 20 and 40 µM) and Apo could signi cantly decrease ROS accumulation in PA-induced HK-2 cells ( Fig. 4C and D, P < 0.01). The results suggested that AS-IV treatment could decrease ROS accumulation in PA-induced HK-2 cells.

AS-IV decreases GSH, SOD expressions in PA-induced HK-2 cells and IL-1β, IL-18 levels in supernatant
To assess the redox state and in ammation in PA-induced HK-2 cells and the protective effect of AS-IV, we further detected the GSH and SOD expressions in HK-2 cells, and the IL-1β, IL-18 levels in supernatant by ELISA. Our results indicated that PA exposure for 6, 9, 12 and 24 h signi cantly decreased the expressions of GSH and SOD in HK-2 cells, and increased the IL-1β and IL-18 levels in supernatant compared with BSA control group (Fig. 5A, B, E and F, P < 0.05 or P < 0.01). On the contrary, after administration with AS-IV and Apo for 24 h, the GSH and SOD expressions were signi cantly increased in HK-2 cells, and theIL-1β and IL-18 levels in supernatant were signi cantly decreased compared with PAinduced model group (Fig. 5C, D, G and H, P < 0.05 or P < 0.01). The results suggested that PA exposure could decrease the antioxidant function and increase in ammation in HK-2 cells, while AS-IV treatment could improve oxidative stress and in ammation in PA-induced HK-2 cells.
3.6. AS-IV downregulates expressions of NLRP3, caspase-1, ASC, IL-1β and NOX4 in PA-induced HK-2 cells NOX4 is an important enzyme contributing to ROS generation, which can further activate NLRP3 in ammasome. We further used immunoblot analysis to con rm whether NOX4 and NLRP3 in ammasome are involved in PA-induced redox imbalance and in ammation in HK-2 cells. The results showed that PA exposure for 6, 9, 12 and 24 h could signi cantly increase the expressions of NLRP3, ASC, caspase-1 and NOX4 in HK-2 cells compared with BSA control group (Fig. 6A-E, P < 0.05 or P < 0.01). AS-IV (10, 20, and 40 µM) and Apo (100 µM) treatment for 24 h signi cantly decreased the expressions of NLRP3, caspase-1, ASC and NOX4 in HK-2 cells compared with PA-induced model group (Fig. 6F-J, P < 0.05 or P < 0.01). These data suggested that NOX4 and NLRP3 in ammasome activation closely involved in PA-induced HK-2 cells injury, and AS-IV treatment could inhibit NOX4 and NLRP3 in ammasome activation and protect against PA-induced HK-2 cells damage.
To further con rm the protective effect of AS-IV on PA-induced NLRP3 in ammasome activation, we also examined the expression of NLRP3 by immuno uorescence. The results indicated that PA stimulation signi cantly increased the expression of NLRP3 in HK-2 cells compared with BSA control group. Meanwhile, AS-IV and Apo treatment could decrease NLRP3 expression compared with model group (Fig. 7A and B, P < 0.01).

AS-IV alleviates renal histopathology and lipid deposition in T2DM rats
To con rm the effect of AS-IV on kidney tubular injury, we examined the renal histopathology and lipid deposition in T2DM rats. The HE staining results indicated that the renal tubules showed signi cant vacuolar degeneration and enlarged lumenin in the T2DM model group as compared with control group. And AS-IV (80 mg/kg) and Met (200 mg/kg) treatment for 8 weeks signi cantly improve these pathological changes as compared with model group (Fig. 8A). In addition, Oil red O staining was used to detect the lipid deposition in the kidney. As shown in Fig. 8B and C, the lipid deposition was signi cantly increased in the T2DM model rats as compared with the control group. And AS-IV (80 mg/kg) and Met (200 mg/kg) treatment for 8 weeks signi cantly decreased the lipid deposition as compared with model group. The data suggested that AS-IV treatment could alleviate the renal tubular histopathology and lipid deposition in T2DM rats.

AS-IV downregulates NLRP3 and NOX4 expressions in renal cortex of T2DM rats
To further assess the effect of AS-IV treatment on NLRP3 in ammasome and NOX4 activation in T2DM rats, we detected the expressions of NLRP3 and NOX4 related proteins in the renal cortex by using immunoblotting. The results showed that the expressions of NLRP3, caspase-1, ASC, IL-1β, NOX4, p22phox, and p47phox were signi cantly increased in the T2DM model rats compared with control group (Fig. 9, P < 0.01). And AS-IV (80 mg/kg) and Met (200 mg/kg) treatment for 8 weeks signi cantly decreased the expressions of NLRP3, caspase-1, ASC, IL-1β, NOX4, p22phox and p47phox in renal cortex of the T2DM rats compared with model group (Fig. 9, P < 0.05 or P < 0.01).
To further demonstrate the effect of AS-IV on NLRP3 and NOX4 expressions in renal tubules, the immunohistochemistry was performed to detect the expressions of NLRP3 and NOX4. The results indicated that the expressions of NLRP3 and NOX4 were signi cantly increased in the tubules in the T2DM model group compared with control group (Fig. 10, P < 0.01). Meanwhile, compared with model group, AS-IV (80 mg/kg) and Met (200 mg/kg) treatment could signi cantly decrease the expressions of NLRP3 and NOX4 in the tubules (Fig. 10, P < 0.05 or P < 0.01). The data suggested that the activation of NOX4 and NLRP3 in ammasome might play important roles in the progression of DN, and AS-IV treatment could alleviate renal tubular injury via inhibiting NOX4 and NLRP3 in ammasome activation in the DN.

Discussion
Tubular epithelial cells damage was commonly reported as an important element in DN [19]. Meanwhile, high levels of FFAs and renal tubular damage were observed in T2DM patients and model rats [24], suggesting that excessive FFAs might play an important role of in renal tubular injury in DN. In this study, our results suggested that PA exposure could signi cantly exacerbate renal tubular epithelial cells damage, which were also found in the T2DM rats. However, AS-IV treatment signi cantly attenuated renal tubular damage in vitro and in vivo. Meanwhile, the results also indicated that NOX4 and NLRP3 in ammasome were signi cantly increased in PA-induced HK-2 cells and in renal tubules of T2DM rats, while AS-IV treatment signi cantly inhibited the expressions of NOX4 and NLRP3 in ammasome in vitro and in vivo. These data suggested that AS-IV treatment could protect against excessive FFAs-induced renal tubules damage via inhibiting the NOX4 and NLRP3 in ammasome activation.
Excessive FFAs-induced lipids deposition in tissues is one of the most important factors that induce oxidative stress and in ammation in various tissues and organs of the body [25][26][27]. PA is an important member of the saturated fatty acid family and closely involves in the occurrence and development of a lot of diseases in the body, such as insulin resistance and T2DM [28][29][30]. When the body's lipids are overloaded, the lipid balance is disrupted, leading to lipid metabolism system unable to proceed normally and cause lipotoxicity [31,32]. It has been reported that cellular lipotoxicity contributes to obesity-related nephropathy [33]. The main saturated fatty acids, such as PA, has shown contributions to renal damage through increasing excessive ROS generation, apoptosis and endoplasmic reticulum (ER) stress [34,35]. In our previous studies, obvious renal lipid deposition and tubules damages were found in PA-induced HK-2 cells and T2DM rats [33,36]. In this study, we found that, with the prolongation of PA exposure, the cells viability was signi cantly decreased, and the apoptosis and lipids deposition were also signi cantly increased in HK-2 cells. However, AS-IV treatment signi cantly increased the cells viability, and decreased the apoptosis and lipids deposition in PA-induced HK-2 cells. And the in vivo study also suggested that AS-IV treatment could signi cantly attenuate the lipids deposition in tubules and ameliorate the pathological injury of renal tubules in T2DM rats. The data suggest that AS-IV has protective effect on PA-induced renal tubular damages.
At present, the potential molecular mechanisms of excessive FFA-induced renal tubular damages still remain unclear. Oxidative stress and in ammation have been reported to play important roles in the evolution of DN [37]. Oxidative stress occurs when the excessive generation of ROS overloads the ability of the organic antioxidant system and causes damage to DNA, proteins and lipid [38]. Excessive ROS generation has been characterized in DN, and impaired antioxidant defense system is also observed in obesity-associated metabolic syndrome [39][40][41]. In recent years, growing studies have indicated that NADPH oxidase (NOX) is an important source of ROS generation in body [42]. The NOX is composed of a membrane subunit (NOX1-5) and catalytic subunits of p47phox, p22phox, p67phox. Growing studies con rmed that NOX4 is widely expressed in renal tubular epithelial cells, and abnormal expression of NOX4 is closely related to renal tubular damages [43,44]. It has been reported that glycosylated albumin can induce the up-regulation of NOX4 expression, leading to brosis and apoptosis in NRK-52E cells, and promoting the development of DN [45]. However, it is still unknown whether NOX4 is involved in PAinduced renal tubular damages. GSH and SOD play important roles in keeping balance of oxidation and antioxidant capacity in the body, and the amount of them can also re ect the body's antioxidant capacity [46]. Previous studies showed that AS-IV signi cantly inhibited high glucose-induced apoptosis of HK-2 cell via increasing the antioxidant enzymes activities of GSH, SOD and CAT, and reduced the high glucose-induced ROS generation in HK-2 cells [1]. In the present study, our results suggested that PA exposure signi cantly increased ROS production and NOX4 expression, and decreased the GSH and SOD levels in HK-2 cells. Meanwhile, our results also indicated that the expressions of NOX4, phox22 and phox47 were signi cantly increased in the renal tubules in T2DM rats. Apocynin (Apo) is often used as a NOX inhibitor, which can inhibit the activity of NOX by interfering with the intracellular translocation of p47phox and p67phox [47]. The present results showed that both AS-IV and Apo treatment could signi cantly decrease the ROS accumulation, NOX4 expression and increase the levels of GSH and SOD in PA-induced HK-2 cells. Meanwhile, AS-IV treatment also reduced the expressions of NOX4, phox22 and phox47 in the renal tubules in T2DM rats. These data suggested that AS-IV could attenuate PA-induced ROS oxidative stress by inhibiting NOX4 expression in renal tubular epithelial cells.
In ammation is also an important cause for renal tubules damages contributing to DN. In recent years, increasing data have characterized the diabetic renal brosis as a chronic in ammatory response [48]. For example, DN is accompanied by in ltration of in ammatory cells, up-regulation of in ammatory cytokines and in ammatory responses in the kidney [49]. Inhibition of in ammatory responses has been reported to have protective effect on experimental diabetic kidney disease [50]. In ammasomes play a central role in the in ammatory response, and activation of in ammasomes can cleave the procaspase-1 to caspase-1, eventually increasing the generation of proin ammatory factors, such as IL-1β and IL-18 [15]. The NLRP3 is a core of the in ammasome, and has been reported to be involved in the progression of renal brosis and contribute to the pathogenesis of chronic kidney disease and DN [17]. Previous research showed that the expressions of NLRP3, caspase-1, along with the maturation of IL-1β and IL-18 were signi cantly increased in high glucose-induced HK-2 cells [51]. Also, it has been reported that accumulation of ROS contributes to HG-induced activation of NLRP3 in ammasome in epithelial cells [52]. In addition, studies have shown that AS-IV can inhibit NLRP3 in ammasome activation and the proin ammatory cytokines expression to play an anti-in ammatory effect by improving antioxidant capacity, resulting in attenuation of lipopolysaccharide-induced acute kidney injury [53][54][55]. Our preliminary experiments also indicated that AS-IV could signi cantly reduce HK-2 cells injury and apoptosis [22]. However, it is still not completely understood whether AS-IV protects against PA-induced renal tubular injury by regulating NOX4-NLPR3 in ammasome. In this work, the results showed that the expressions of NLRP3, ASC, caspase-1, and pro-in ammatory factors of IL-1β and IL-18 were signi cantly increased in PA-induced HK-2 cells and in renal tubules of T2DM rats. After AS-IV and Apo treatment, the expressions of NLRP3, ASC, caspase-1, IL-1β and IL-18 were signi cantly decreased in PA-induced HK-2 cells.
Meanwhile, the results in vivo also indicated that AS-IV and Met treatment could signi cantly downregulate the expressions of NLRP3, ASC, caspase-1 and IL-1β in renal cortex of T2DM rats. The data suggested that PA exposure might mediate the in ammatory response by activating the NLRP3 in ammasome in DN. And AS-IV treatment might alleviate the PA-induced renal tubular damages through inhibiting the NLRP3 in ammasome expression.

Conclusion
In general, our study indicated that AS-IV treatment alleviated renal tubular epithelial cells injury both in PA-induced HK-2 cells and in renal tubules of T2DM rat, and the mechanism may be related to inhibition of NOX4 and NLRP3 in ammasome, which attenuates oxidative stress and in ammation. However, this study only provided the protective effect of AS-IV on PA-induced HK-2 cells, the effect and mechanism of high glucose and hyperlipidemia on renal tubular epithelial cells remain for further study. Nevertheless, this study provides evidence for the protective effect of AS-IV on PA-induced renal tubular epithelial cells damage. Furthermore, regulating NOX4 and NLRP3 in ammasome may be an effective strategy to improve renal tubules damage in T2DM.