Ginsenoside Rg1 Alleviates Podocyte Injury Induced by Hyperlipidemia via Targeting the mTOR/NF-κB/NLRP3 Axis

Background Podocyte injury plays an important role in diabetic nephropathy (DN). The aim of this study was to determine the potential therapeutic effects of the ginsenoside Rg1 on hyperlipidemia-stressed podocytes and elucidate the underlying mechanisms. Methods In vitro and in vivo models of DN were established as previously described, and the expression levels of relevant markers were analyzed by Western blotting, real-time Polymerase Chain Reaction (PCR), immunofluorescence, and immunohistochemistry. Results Ginsenoside Rg1 alleviated pyroptosis in podocytes cultured under hyperlipidemic conditions, as well as in the renal tissues of diabetic rats, and downregulated the mammalian target of rapamycin (mTOR)/NF-κB pathway. In addition, Rg1 also inhibited hyperlipidemia-induced NLRP3 inflammasome in the podocytes, which was abrogated by the mTOR activator L-leucine (LEU). The antipyroptotic effects of Rg1 manifested as improved renal function in the DN rats. Conclusion Ginsenoside Rg1 protects podocytes from hyperlipidemia-induced damage by inhibiting pyroptosis through the mTOR/NF-κB/NLRP3 axis, indicating a potential therapeutic function in DN.


Introduction
Diabetic nephropathy (DN) is a progressive microvascular complication of diabetes mellitus (DM), and the primary cause of end-stage renal diseases (ESRD) [1]. Although the exact mechanism is unclear, several studies have implicated podocyte injury as the key etiological factor [2][3][4]. Podocytes are specialized glomerular epithelial cells that form the outermost filtration layer, and their loss/damage is a hallmark of various reno-pathological conditions including DN [5,6]. Pyroptosis, a type of programmed cell death characterized by swelling, pore formation, and release of proinflammatory cytokines (interleukin-(IL-) 1β and IL-18), is a potential mechanism of podocyte loss. In fact, pyroptosis has been observed during the development of acute kidney disease and DN, although the specific mechanism is unknown [7]. e pyroptotic cascade is triggered by the nucleotide oligomerization domain-(NOD-) like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex consisting of NLRP3, apoptosis-associated speck-like protein (ASC) with caspase recruitment domain (CARD) domain and procaspase-1 [8,9], and the downstream inflammatory pathway is driven by caspase-1 [10,11]. NLRP3 is activated by mTOR, a highly conserved serine/threonine kinase (Ser/ r) that regulates cell growth and proliferation [12], in different in vivo and in vitro models [13,14]. Recent studies show that high glucose and fat levels can activate NLRP3 inflammasomes and caspase-1 triggered pyroptosis [15,16]. e inflammatory mTOR-NLRP3-IL-1β axis is activated during lung injury, and its inhibition can reduce the immunopathological damage in lung tissues [17]. e nuclear factor kappa-B (NF-κB) and mTOR signaling pathways are highly interrelated [18,19]. While overexpression of mTOR activates NF-κB, inhibition of mTOR phosphorylation by rapamycin blocks the AKT/NF-κB pathway [20]. In a previous study, we found that inhibiting NF-κB can protect podocytes and reverse DN by blocking mesangial transdermal differentiation [21]. erefore, we hypothesized that the mTOR-NLRP3-IL-1β axis is a potential target for inhibiting podocyte injury in DN. Ginseng has been used in traditional Chinese medicine formulations for over 2000 years, and has antitransdifferentiation, antioxidation, antiapoptosis, and proautophagy effects [22][23][24]. e pharmacological effects of ginseng are mainly attributed to the ginsenoside Rg1, which can effectively inhibit apoptosis and protect podocytes [25,26]. Rg1 also induces autophagy and inhibits apoptosis by activating the AMPK/mTOR pathway [27]. In this study, we analyzed the role of pyroptosis in DN and the therapeutic effects of Rg1.

Cell Culture.
e conditionally immortalized mouse podocyte cell line BNCC337685 was obtained from BeNa Culture Collection (Beijing, China). e cells were cultured in low glucose DMEM (Genview, Florida, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and recombinant IFN-c (PeproTech, London, UK) at 33°C for proliferation, and without IFN-c at 37°C for more than 7 days for inducing differentiation. e cells were harvested at ∼80% confluency and maintained in serum-free conditions for the different assays.

Proliferation Assay.
e podocytes were seeded in 96well plates in serum-free DMEM at the density of 8 × 10 4 per well and cultured for 24 h. Hyperlipidemia was induced with 100 μM palmitate (PA) in the absence or presence of 0, 10, 25, 50, 75, or 100 μM Rg1 for 48 h [28]. e suitably treated cells were then incubated for an additional 2 h with cellcounting kit-8 (CCK-8, KeyGEN BioTECH, Nanjing, China) solution (10 µL/well), and the optical density of each well was measured at 450 nm. A decrease in cell viability to 30-40% was indicative of podocyte injury [29,30].  [31,32]. e diabetic rats were fed the high fat diet (HFD; 10% lard, 20% sucrose, 2.5% cholesterol, 0.5% sodium cholate, and 67% basic feed) for 4 weeks to induce DN, while the control rats were given normal food. Four weeks after STZ injection, the successfully modelled DN rats were randomly divided into the (untreated) DN and ginsenoside Rg1-treated groups (n � 8 in each group) and administered with vehicle (aqua distillate) or 50 mg/kg ginsenoside Rg1 once daily by oral gavage for 8 weeks. is dose of ginsenoside Rg1 has been proved to be effective and safe [33]. At the end of the regimen, the rats were anaesthetized adequately; then, the urine creatinine (UCr), urinary microalbumin (Malb), blood urea nitrogen (BUN), and serum creatinine (Scr) levels were measured. e rats were then euthanized, and their renal cortices were harvested for further analyses. All animal experiments were approved by the Institutional Animal Care and Use Committee at Capital Medical University and conformed to the Guidelines for the Care and Use of Laboratory Animals by the National Institute of Health. (PAS), and Masson's staining were performed as per standard protocols, and the tissue sections were observed under a light microscope (Leica DM60008, Kyoto, Japan). For transmission electron microscopy (TEM), the renal cortices were fixed in 2% glutaraldehyde (Servicebio, Wuhan, China), cut into ultrathin sections, and stained accordingly [34]. e samples were observed under a TEM (JEM-1400 plus, JEOL, Tokyo, Japan).

Biochemical
Assays. e levels of serum creatinine, urine creatinine, and urine albumin were measured using specific kits from Leadman Biochemistry (Beijing, China) in accordance with the manufacturer's instructions. e biochemical parameters were detected with an automatic chemistry analyzer (7600, HITACHI, Tokyo, Japan). e urinary protein content was calculated relative to the creatinine levels.

TUNEL Assay.
Tissue sections and podocytes cultured on coverslips were treated with proteinase K (20 g/mL) and stained using the In-Situ Cell Death Detection kit (Nanjing KeyGen Biotech Co. Ltd., China) according to the manufacturer's instructions. e number of TUNEL-positive cells was counted in 10 randomly selected, nonoverlapping fields at 200x magnification. Apoptotic index was calculated as the number of TUNEL-positive cells per 10 3 cells and %TUNEL-positive podocytes.

Reverse Transcription Quantitative Polymerase Chain
Reaction (RT-qPCR). Total RNA was isolated from the cultured podocytes and renal cortices using TRIzol Reagent according to the manufacturer's instructions and reversetranscribed using the PrimeScript ™ RT kit. e qRT-PCR analysis was performed using the SYBR ® Premix Ex Taq ™ II (Takara) in the ABI PRISM 7500 FAST Real-TIME PCR System (ABI, Vernon, CA, USA). Relative gene expression levels were calculated by the 2ΔCT method. e sequence of mTOR primers was as follows: forward: 5′-AGT-GAAGCCGAGAGCAATGAGA-3′ and reverse: 5′-GACAAGGAGATAGAACGGAAGAAGC-3′.

Propidium Iodide (PI)/Hoechst 33342 Double Staining.
e suitably treated podocytes were harvested and stained with Hoechst 33342 at 37°C for 10 minutes. e cells were washed with PBS 3 times, and then the 1 × Buffer and the PI were added to stain the podocytes in the dark for 10 minutes. e cells were observed under an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Whether the PI could penetrate the pyroptosis cell membrane and enter into the nucleus was determined by the appearance of red fluorescence, indicating the presence of dead cells as a result of pyroptosis. e pyroptosis rate was calculated as the number of dead cells/the total number of cells × 100%. e experiment was conducted in triplicate.

Statistical Analysis.
e data were presented as mean-± standard deviation (SD). Statistical analysis was performed using the SPSS 19.0 software (IBM Corporation, Armonk, NY, USA). Student's t-test was used to compare the difference between two groups, while multiple groups were compared by one-way analysis of variance (ANOVA). A P value <0.05 was considered statistically significant.
Evidence-Based Complementary and Alternative Medicine 3

Ginsenoside Rg1 Protects Podocytes against Hyperlipidemia-Induced
Injury. e optimal therapeutic dose of Rg1 was determined in an in vitro model of podocyte injury. As shown in Figure 2(a), Rg1 increased the viability of podocytes in a dose-dependent manner at lower concentrations for 48 h, while doses above 50 μM were cytotoxic. erefore, 50 μM Rg1 for 48 h was used for subsequent experiments. Podocytes exposed to hyperlipidemic conditions showed reduced activity and low expression level of nephrin, while these effects were restored by Rg1 treatment (Figures 2(a) and 2(b)). Consistent with the above observation in vitro, in normal group rats, nephrin staining presented a green linear-like pattern along the capillary loops of glomeruli. Instead, a light green, discontinuous, short linear-like or punctiform staining pattern of nephrin was observed in DN group, and Rg1 also upregulated the in situ expression of nephrin in the glomeruli of DN rats (Figure 3). In vitro, actin stress fibers were arranged regularly or parallel to each other in podocytes of normal group. Phalloidin staining further revealed the collapsed or totally absent intracellular actin stress fibers in the hyperlipidemiatreated podocytes, resulting in the polygonal cells. Ginsenoside Rg1 treatment remodeled the podocyte skeleton and restored their normal shape (Figure 2(c)).
Taken together, Rg1 protects podocytes against hyperlipidemia-induced injury by restoring nephrin levels and the cytoskeleton.

Ginsenoside Rg1 Alleviated Hyperlipidemia-Induced Pyroptosis in Podocytes.
e positive rate of PI staining revealed that, compared with the NC group, there was significant increased pyroptosis rate in PA-induced podocytes (Figures 4(a) and 4(b)). Pyroptosis is also characterized by caspase-1 activation and IL-1β secretion [35]. As shown in Figures 4(c) and 4(d), the expression of Casp1 p20 and IL-1β was markedly increased in the hyperlipidemia-stressed podocytes. Consistent with this, pretreatment of the hyperlipidemiastressed podocytes with 20 μM caspase-1 inhibitor z-YVAD-FMK significantly increased cell viability and nephrin levels, decreased the rate of PI-positive podocytes, and restored the cytoskeletal structure (Figures 2(a)-2(c), 4(a) and 4(b)), thus further confirming that hyperlipidemia-induced podocyte injury in vitro was partly mediated via pyroptosis induction. Furthermore, Rg1 significantly decreased pyroptosis rates in the suitably treated podocytes, which was also correlated to markedly lower levels of Casp1 p20 and IL-1β compared to the PA group (Figures 4(a)-4(d)). However, the pyroptosis rate and the level of Casp1 p20 of Rg1 group were higher than those in the PA + z-YVAD-FMK groups; these results also strongly suggested that hyperlipidemia induced pyroptosis of podocytes, which was alleviated by Rg1.
Moreover, increased TUNEL-positive cells were observed in the glomeruli and renal tubules of the DN rats, which was significantly alleviated by Rg1 treatment (Figures 5(a) and 5(b)). Immunofluorescence images and immunoblot showed that the glomeruli of the untreated DN rats also expressed higher levels of activated caspase-1 (Casp1 p20) and IL-1β, which was decreased by Rg1 (Figures 5(c) and 5(d)).

Ginsenoside Rg1 Targets the mTOR/NF-κB Pathway in
Podocytes under Hyperlipidemic Conditions. Studies have implicated mTOR in podocytes injury and DN [36,37]. Consistent with this, hyperlipidemia treatment significantly increased total and phosphorylated mTOR levels in the cultured podocytes (Figures 6(a) and 6(b)), as well as in renal tissues of diabetic rats (Figure 7). Furthermore, pretreatment with the mTOR inhibitor rapamycin (10 ng/mL) decreased total and phosphorylated mTOR levels (Figures 6(a) and 6(b)), inhibited the expression of Casp1 p20 and IL-1β, and also abrogated hyperlipidemia-induced pyroptosis (Figure 4). Rg1 significantly downregulated mTOR and p-mTOR (Figure 6(a)), as well as p-p65 (Figure 6(c)) in the hyperlipidemic podocytes. However, pretreatment of these podocytes with the mTOR activator LEU (5 mM) abrogated the suppressive effects of Rg1 on p-p65 (Figure 6(c)). erefore, the mTOR/NF-κB pathway may be one of the effect targets of Rg1 on the PA-induced podocytes.
Similarly, mTOR and p-p65 were significantly upregulated in the renal tissues of untreated DN rats compared to the NC group and decreased following Rg1 treatment (Figure 7).  Values are the mean ± SD; n � 3. a P < 0.05 compared to the NC group, b P < 0.05 compared to the PA group, c P < 0.05 compared to the PA + Rg1 group. Abbreviations: Rapa, rapamycin.

Rg1 Inhibited Hyperlipidemia-Induced NLRP3 in Podocytes by Suppressing the mTOR/NF-κB Pathway.
Since several inflammasomes can trigger pyroptosis, we silenced NLRP3 in the podocytes in order to determine whether NLRP3 inflammasome is essential for hyperlipidemia-induced pyroptosis in podocytes. As shown in Figure 8, NLRP3 knockdown (Figure 8(a)) significantly reduced the pyroptosis rate (Figure 8(c)) and the expression of Casp1 p20 and IL-1β in the podocytes exposed to hyperlipidemic conditions (Figure 8(b)). is suggests that podocyte pyroptosis induced by hyperlipidemia was mainly regulated by NLRP3 inflammasome.
Previous studies have shown that NLRP3 is activated by mTOR/NF-κB [13,38]. Consistent with this, the NF-κB inhibitor JSH-23 (20 μM) and rapamycin significantly decreased the expression of ASC and Casp1 p20 in the hyperlipidemic podocytes (Figure 9), which were significantly elevated by LEU.
Interestingly, JSH-23 neutralized the activation effects of LEU on NLRP3 under hyperlipidemic conditions (Figure 9). Furthermore, Rg1 markedly inhibited the expression and activation of NLRP3 under hyperlipidemic conditions; however, the inhibitory effect of Rg1 on NLRP3 was attenuated by LEU ( Figure 9). NLRP3, ASC, and Casp1 p20 levels were also upregulated in the glomeruli of DN rats and deceased upon Rg1 treatment (Figures 5(c)-5(e)). ese findings indicated that hyperlipidemia-induced NLRP3 activation was dependent on the mTOR/NF-κB pathway, and Rg1 targeted the mTOR/NF-κB/NLRP3 axis to alleviate pyroptosis in the podocytes.

Effect of Ginsenoside Rg1 on Renal Function and Morphology in Diabetic Rats.
e therapeutic effects of ginsenoside Rg1 on renal function and morphology were measured in terms of standard biochemical and histopathological indices. Rg1   Values are the mean ± SD; n � 3. a P < 0.05 compared with the NC + si-con group, b P < 0.05 compared with the PA + si-con group, c P < 0.05 compared with the NC + si-NLRP3 group. Abbreviations: si-NLRP3, the podocytes transfected by the NLRP3-specific siRNA; si-con, the podocytes transfected by scrambled siRNA.

Discussion
e ginsenoside Rg1 is one of the pharmacologically active components of ginseng and exhibits antiapoptotic, antiinflammatory, and neuroprotective effects [39][40][41][42]. It also has a significant reno-protective action [43] and alleviates aldosterone-induced podocyte injury [44]. In line with these findings, we found that Rg1 also protected podocytes from hyperlipidemic injury both in vivo and in vitro.
Pyroptosis is a form of programmed cell death mediated by the NLRP3 inflammasome and caspase-1 and is the pathological basis of various inflammatory and degenerative disorders [17,35]. It is frequently induced by hyperlipidemia and therefore associated with diabetic cardiomyopathy and diabetic retinopathy [45,46]. Recently, Wang et al. detected increased pyroptosis in the tubular cells involved in diabetic kidney disease (DKD) [7]. Similarly, we also observed pyroptosis in podocytes exposed to hyperlipidemic conditions, indicating its involvement in DN. e mTOR pathway has been previously implicated in podocyte injury [47] and was also upregulated in the affected cells in our study. In addition, there is a positive crosstalk between mTOR and NF-κB [48,49], and Wei et al. associated NF-κB pathway activation with damaged podocytes [50]. Rg1 significantly inhibited pyroptosis, restored nephrin expression and cytoskeletal structure in the podocytes, and blocked hyperlipidemia-induced mTOR/NF-κB signaling. e pyroptotic cascade is triggered by the NLRP3 inflammasome, which then activates caspase-1 and releases IL-1β [51][52][53]. While IL-1β mediates podocyte injury and renal inflammation [54], NLRP3 is involved in the pathogenesis of chronic kidney disease [55,56]. Since hyperlipidemia can activate multiple inflammasomes [57], we knocked down NLRP3 in podocytes and found that absence of NLRP3 inhibited pyroptosis, indicating that it initiates pyroptosis in podocytes during DN.
treated with LEU showed high levels of NLRP3, ASC, and Casp1 p20. In addition, JSH-23 abrogated the effect of LEU on the activation of NLRP3, indicating that mTOR activates NLRP3 via NF-κB. Rg1 also significantly decreased NLRP3 levels in the hyperlipidemia-exposed podocytes. Furthermore, the inhibitory effects of Rg1 on the activation of NLRP3 were reversed by LEU, which strongly suggested that Rg1 decreased hyperlipidemia-induced pyroptosis in podocytes by inhibiting the mTOR/NF-κB/NLRP3 axis. A previous study showed that the combination of Rg1 and astragaloside relieved renal fibrosis in DN by inhibiting the TGF-β1/Smads pathway and oxidative stress [33]. Consistent with this, Rg1 significantly reduced proteinuria, improved renal function, and alleviated tissue damage in diabetic rats. Podocyte is the functional unit of the glomerular filtration membrane [61], and its injury is the pathological basis of DN [62]. us, Rg1-mediated podocyte rescue translated to improved renal function. In summary, Rg1 improves renal function in DN rats by inhibiting hyperlipidemia-induced pyroptosis in podocytes via targeting mTOR/NF-κB/NLRP3 signaling. Our findings provide the experimental basis for future clinical trials on Rg1 as a treatment option for DN.

Conclusions
Ginsenoside Rg1 protects podocytes from hyperlipidemiainduced damage by inhibiting pyroptosis through the mTOR/NF-κB/NLRP3 axis, indicating a potential therapeutic function in DN.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.