STZ inhibitor

Silymarin nanoliposomes attenuate renal injury on diabetic nephropathy rats via co-suppressing TGF-β/Smad and JAK2/STAT3/SOCS1 pathway

Yi Chen a, Li Chen b, Taiwang Yang c,*

Abstract

Aims: To investigate the improvement and mechanisms of silymarin on renal injury in mouse podocytes and streptozotocin (STZ)-induced diabetic nephropathy model (DN) rats.
Main methods: Firstly, the effects of silymarin on the cell viability and cellular injury-related indicators of high- glucose incubated mouse podocytes MPC-5 were assessed by CCK-8 and western blotting (WB) methods, respectively. The STZ-induced diabetic rats with DN were treated with silymarin nanoliposomes at three doses for consecutive 8-week. General metabolic indicators, renal functions and lipid accumulation-related factors were all measured. The renal tissue sections were stained and observed via hematoxylin-eosin (H&E) staining method. Real-time RT-PCR and WB methods were utilized to measure the expression of JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathway related factors.
Key findings: Silymarin significantly improve the high-glucose induced up-regulation of podoxin and nephrin, as well as the expression of inflammatory cytokines IL-6, ICAM-1 and TNF-α, and the cell survival rates were also significantly increased in a dose-dependent manner. Significant improvement on body weight/kidney ratio, renal functions and lipid profiles in renal tissues were observed in STZ-induced diabetic rats after chronic silymarin treatment. The H&E staining exhibited that the pathological damages in renal tissues were obviously improved. Moreover, silymarin nanoliposomes treatment notably suppressed expression levels of inflammation-related proteins as well as IL-6 and ICAM-1, and regulated JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathway, thereby exhibited protective effects on kidney of DN model rats.
Significance: Silymarin nanoliposomes ameliorate STZ-induced kidney injury by improving oxidative stress, renal fibrosis, and co-inhibiting JAK2/STAT3/SOCS1 and TGF-β/Smad signaling pathways in diabetic rats.

Keywords:
Silymarin nanoliposomes
Kidney injury
Oxidative stress
Inflammation
JAK2/STAT3/SOCS1
TGF-β/Smad

1. Introduction

Diabetes mellitus (DM) is a global epidemic with high morbidity and mortality [1,2]. Except for the neuropathic lesions and cardiovascular complications, DM is associated with kidney-related mortality with increased risk of renal injury and failure [3,4]. Diabetic kidney disease (DKD) is a common cause of kidney disease with prevalence rate being up to 70% in diabetic patients [4,5]. Diabetes and obesity predispose to abnormal oxidative stress in renal tissues, which leads to renal dysfunction and abnormal metabolism [5–7]. DKD covers from simple oxidative stress to irreversible fibrosis with varying degrees of inflammation and oxidative stress damage [8–10]. However, the pathogenesis of early DKD is very complex [6]. During the development and progression of DKD, extracellular matrix deposition, glomerular dilatation, basement membrane thickening, and podocyte loss occur simultaneously or continuously [4,9]. At the same time, the appearance of urinary microalbumin (UMA) is a significant warning of early DKD [11]. Although the pathological mechanism of DKD has not been fully elucidated, studies have revealed that oxidative stress and the development of inflammation are both main factors contributing to the development of DKD [12,13]. At present, oral chemical hypoglycemic agents for the treatment of diabetes have certain adverse reactions and damaging effects on the kidney, making the treatment of diabetic renal injury more difficult [14,15].
At present, the traditional drugs were used to treat diabetes and its complications can cause serious adverse reactions, so it is necessary to explore new therapeutic interventions. Recently, the Chinese medicine (TCM) are applied for treating various diseases due to their outstanding biological activity [16,17]. Active ingredients derived from TCM are attracting substantial attention largely due to their safety, less side effects, and comprehensive efficacy [17]. Silymarin is a flavonoid lignan compound extracted from the coat of Silybum marianum seeds, a medicinal plant of the Compositae family, which is widely used in the treatment of clinical liver diseases because of its anti-inflammatory, antioxidant and cell regeneration effects. Recent studies have shown that silymarin also has preventive and therapeutic effects on diabetes and anti-tubulointerstitial fibrosis. In recent decades, more and more attention has been paid to the role of silymarin in DN domestic and overseas, but its specific mechanism of action is not very clear. Sheela et al. used STZ and nicotinamide induction to prepare a rat model of DN, the blood glucose, glycosylated hemoglobin and urinary albumin levels were significantly reduced in rats receiving silymarin treatment, and histopathological assessment showed reduced pathological damage [18].
However, whether the chronic treatment of silymarin could ameliorate the diabetic renal injury is largely unknown, and the molecular mechanism remains to be elucidated. Here, we explored the protective efficacies and the possible mechanism of silymarin treatment on mouse podocytes MPC-5 with oxidative injury and hepatic damage associated oxidative stress and inflammatory indicators induced by STZ injection and high fat feeding in diabetic rats.

2. Material and methods

2.1. Materials

Silymarin were brought from Shanghai Angbo Bio-technology Co., Ltd. (Shanghai, China) with purity >99%. The ELISA kits of lipid peroxidation/malondialdehyde (MDA), serum superoxide dismutase (SOD), catalase, IL-6, IL-8, IL-10, Tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were brought from Sangon Biotechnology (Shanghai, China). All PCR kits and other reagents were obtained from Thermo Fisher Scientific (San Diego, USA). The MPC-5 cells were purchased from the American ATCC Cell Bank. 0.25% EDTA-containing trypsin, DMEM, fetal bovine serum, and CCK-8 kits were obtained from Hyclone (USA).

2.2. Cell culture and assays

MPC-5 cells were cultured in low-glucose DMEM medium in a 37 ◦C, 5% CO2 incubator, and the culture medium was changed every 48 h to 72 h. When the cells were in good condition and fused to about 80%, they were digested and seeded in 96-well plate, 24-well plate or 6-well culture plate to continue the culture process. When the cells were confluent to about 80% with good growth condition, they were given serum-free DMEM medium for 6 h for synchronization and used to the experiments. MPC-5 cells were cultured in high-glucose DMEM medium containing 30 mmol/L glucose to simulate the in vivo environment during diabetes and establish a DN cell model. The normal MPC-5 cells were divided into eight groups under the treatment of 0, 1, 2, 4, 8, 16, 32, 64, 128 μg/mL of silymarin, respectively. And using the untreated cells as normal control. Moreover, the high-glucose incubated MPC-5 cells were divided five groups: (1) Normal cell group (treated with PBS); (2) High glucose group (30 mmol/mL glucose only); (3) High glucose+ low silymarin group (30 mmol/mL glucose+16 μg/mL silymarin); (4) High glucose+ middle silymarin group (30 mmol/mL glucose+32 μg/mL silymarin); (5) High glucose+ high silymarin group (30 mmol/mL glucose+64 μg/mL silymarin). After the cells were treated according to the loading content of each group, they were cultured and incubated in a 37 ◦C, 5% CO2 incubator for 12 h. The cell survival rate was measured with a CCK-8 kit. 20 μL of CCK-8 solution was added to each well and incubated in the incubator for 1 h. The OD values were measured at a wavelength of 450 nm on a microplate reader. The wells with corresponding amount of cell culture medium and CCK-8 solution, but none cells were used as blank control. The relative expressions of podocin, nephrin, TNF-α, IL-6 and ICAM-1 mRNA in the cell supernatant were determined by RT-qPCR methods as previously reported [19].

2.3. Establishment of STZ-induced diabetic rats

Eighty healthy Sprague-Dawley (SD) rats (half male and half female, 8–12 weeks, weighting 170-200 g) were obtained from Guangdong Animal Experimental Center. For one week of acclimatization, all animals were maintained in an SPF grade environment as well as a controlled photoperiod (12 h light/dark cycle). Subsequently, sixty rats feeding with high-fat diet for 4 weeks received the dose of 30 mg/kg STZ after overnight fasting, while the remained 15 rats were fed with conventional diet and treat with citrate buffer (termed Normal group). All STZ-induced diabetic rats were daily treated with different doses of silymarin for 8 weeks and divided into four groups: (1) Diabetic group (treated with 10% DMSO); (2) Low silymarin group (100 μg/kg silymarin); (3) Medium silymarin group (300 μg/kg silymarin); (4) High silymarin group (900 μg/kg silymarin) and using the healthy rats as normal control. All animal experiments were performed in accordance with the institutional guidelines.

2.4. Biochemical analysis

At 9th weeks, all the model rats were sacrificed and the kidney and blood samples were collected. Serum levels of ICAM-1, IL-6, IL-8, IL-10, TNF-α and TGF-β were detected by enzyme-linked immunosorbent assay (ELISA) kit. Kidney homogenates were prepared according to the previously reported procedure, and the levels of MDA, SOD, Catalase and GSH were determined by corresponding assay kits.

2.5. Real-time RT-PCR

The primers used in the following experiments were synthesized from Sangon Biotechnology (Shanghai, China). Total RNA from Kidney tissue was isolated with Qiagen RNeasy mini kit following the manufacturer’s instructions. RNA after RQ1 RNase-free DNase treatment was reverse transcribed into cDNA using high-capacity cDNA reverse transcription kit. Subsequently, real-time RT-PCR was performed using TaqMan Fast Advanced Master Mix on an ABI Fast 7500 system. The amount of mRNA was normalized with an internal reference GBPDH.

2.6. Western blot analysis

Kidney tissue from the rat in each group was lysed by using RIPA solution and further centrifuged at 12,000 rpm at 4 ◦C for 0.5 h. Then the total protein level of supernatant was quantified by using BCA method. Equal amounts of protein from each sample were subjected to SDS-PAGE and then transferred to the PVDF membrane. The gels blocking was performed with 5% nonfat dry milk in TBST at 25 ◦C for 2 h, followed by incubation with primary antibodies overnight at 4 ◦C. After washing with 1 × TBST solution for three times, membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. After washing with 1× TBST solution for three times, specific band was measured using the ECL method via a Bio-Spectrum gel imaging system. Densitometric analysis of the bands was performed with Image J software using the content of β-actin as an internal reference.

2.7. Histopathological evaluations

Part of the tissue from left kidney lobe was fixed in 4% paraformaldehyde for 24 h, then dehydrated and embedded in paraffin to make 5 μm sections. Kidney tissue sections were stained with hematoxylin-eosin (HE) reagent according to standard procedures in the instructions.

2.8. Data analysis

At present study, the one-way analysis of variance (ANOVA) was utilized for the analysis of data. All the results were showed as mean ± SD. The value of p < 0.05 demonstrated the statistically different result. 3. Results 3.1. Silymarin improves the cell viability of MPC-5 cell with oxidative injury induced by high glucose To explore the toxicity and effects of different final concentrations of silymarin on MPC-5 cells, the cellular viabilities were assessed using CCK-8 method. As shown in the Fig. 1, the cellular viabilities of MPC-5 cells did not exhibit obvious decrease under the incubation of increased concentrations of silymarin until 128 μg/mL. Moreover, co-incubation of silymarin at the final concentrations of 16, 32 and 64 μg/mL significantly reversed the decreased cell viability of MPC-5 induced by 30 mmol/mL high-glucose in a dose-dependent manner. Above data collectively indicated that the incubation of silymarin within 16 to 64 μg/mL not only not obviously affected the cell viability but also could protect the MPC-5 cells from the damage induced by high-glucose. 3.2. Incubation of silymarin improves the expression of podocyte-specific proteins and inflammatory factors Compared with podocytes in normal rat group, relative expression of mRNA of podocin and nephrin in the supernatant of podocytes from high-glucose incubation group were notably decreased (both p < 0.05). Significantly, in the three silymarin-treated podocytes, the relative expression of podocin and nephrin mRNA were both significantly improved compare to podocytes in high-glucose group (all p < 0.05). In addition, the relative expression of ICAM-1, IL-6 and TNF-α mRNA in the supernatant of podocytes were obviously up-regulated in high-glucose alone group (all p < 0.01) compare to the normal MPC-5 cell group, suggesting that podocyte culture in the high glucose environment will produce a significant inflammatory response, and then reduce the cell survival rate. In addition, the relative expression of ICAM-1, IL-6 and TNF-α mRNA in podocytes were notably improved in silymarin group compare to podocytes in high-glucose group (all p <0.05), but there was no significant dose-dependent relationship between the incubation doses (Fig. 2). 3.3. Chronic treatment of silymarin improves metabolic characters and renal functional indicators of DN model rats We further assessed the protective effects of chronic treatment of silymarin at different doses on DN model rats. Firstly, diabetic nephropathy is usually accompanied by abnormally larger renal tissue, while the kidney indexes in 300 and 900 μg/kg doses of silymarin treated DN model rats were both well maintained and obviously lower than that of saline-treated DN ones. Significantly, the excessive BGLs of DN model rats were reversed after chronic treatment of silymarin at all three dosages (100, 300 and 900 μg/kg), of which the difference was both significant compare to those of the saline-treated ones. Moreover, kidney functional indicators of DN model rats, including 24 h-uninary proteins, BUN and SCr levels, were all significantly evaluated compared with those of the normal rats. As the results showed in the Fig. 3, the ratio of kidney/body weight and FBGLs of the DKD rats were significantly increased compare to those of the normal rats. Surprisingly, the DN model rats received silymarin at all three doses exhibited obviously normalized renal function compared with those of the saline-treated DN model rats (all p < 0.05). All the above results collectively demonstrated long-term administration of silymarin could effectively improve diabetes and DN related metabolic disorders in the DN model rats. 3.4. Chronic treatment of silymarin ameliorated kidney fibrosis in DN model rats We further assessed protective efficacies of silymarin at three doses on histopathological changes of renal cells of model rat with DN under the chronic treatment. As shown in Fig. 4A, the healthy rats showed normal state of glomeruli and matrix, and there was no obvious abnormal enlargement of glomerular cells. However, the model rats in DN group showed more blurred glomerular capillary structure, widened and increased matrix, a certain degree of sclerosis or complete sclerosis, and severe fibrotic lesions occurred. After long-term three doses of silymarin (100, 300 and 900 μg/kg) intervention, the glomerular vascular structure of DN model rats was significantly improved compared with the placebo group, the glomerular structure was also widened, some glomeruli returned to normal, and fibrosis was alleviated. We furtherly detected the expression levels of the fibrosis-related indicators in renal tissues to explain the chronic effects of silymarin against renal fibrosis. According to the results shown in the Fig. 4, the protein expression levels of TGF-β1, Collegen-1 and fibronectin were analyzed using WB methods and data indicated significantly up- regulated or down-regulated in saline or different doses of silymarin treated groups and the difference were all significant (all p < 0.05). Above results collectively demonstrated that 8-week administration of silymarin at all three doses could improve renal fibrosis suppressing the expression levels of fibrosis-related factors in DN model rats. 3.5. Effects of silymarin on oxidative stress, lipid peroxidation and antioxidant enzyme system In order to clarify the efficacies of silymarin administration on lipid peroxidation in kidney tissues of DN model rats, we detected the expression levels of MDA and results showed that the MDA was notably upregulated in DN model rats compare to normal ones. Significantly, the obviously increased content of MDA was reversed in response to silymarin treatment at all three doses. In addition, we also investigated the effects of silymarin at three doses on the activity of key anti-oxidant enzymes, catalase, GSH-Px, and SOD in kidney of DN model rats. As the results showed in Fig. 5, activities of GSH-Px, SOD, as well as catalase were greatly inhibited in kidney tissues of the DN model rats. Significantly, chronic treatment of all three doses of silymarin could effectively improve the altered activities of catalase, SOD as well as GSH- Px in the kidneys of DN model rats. Therefore, above data collectively demonstrated that silymarin held potent ability to inhibit oxidative stress by enhancing antioxidant enzyme systems and further to ameliorate kidney injuries. 3.6. Chronic treatment of silymarin ameliorated renal injury by regulating the JAK2/STAT3/SOCS1and TGF-β/Smad signaling pathway In present research, relative expression of inflammatory factors, including IL-6, IL-8, TNF-α, IL-10, TGF-β and ICAM-1 in kidney were all detected. As illuminated in Fig. 6, all the protein expression levels of above-mentioned factors were significantly increased in the DN model rats compared with those of the healthy normal rats. Significantly, chronic treatment of silymarin effectively down-regulated the content of these inflammatory indicators, especially for IL-6 and ICAM-1 almost in a clearly dose-dependent manner, in renal tissues of DN model rat, suggesting the inflammatory factors, which mediates the kidney injuries in DN model rats, could be effectively improved under chronic treatment of silymarin. Considering that notably up-regulation of inflammatory factors could be induced by the activation of JAK/STAT signal pathway, we furtherly explored the relative protein expression levels of its related indicators, and the WB results exhibited that the protein levels of p- STAT3 and p-JAK2, or SOCS1, were notably up-regulated or down- regulated, respectively, in renal tissues of DN rats compare to those of healthy normal rats (Fig. 7, all p < 0.05). In contrast, the expression of p- STAT3 and p-JAK2 in silymarin treated groups were all obviously decreased, while the expression of SOCS1 was obviously increased (all p < 0.05). Previous studies have found that TGF-β1 plays a very important role in the development and progression of DN. TGF-β1 is an essential membrane-bound protein for the TGF-β signaling pathway, and Smads mediate signaling in the TGF-β signaling pathway. Therefore, we furtherly detected expression levels of TGF-β/Smad signaling pathway- related factors, including Smad6/7, Smad2/3 and TGF-β1, in kidney lysates from DN model rat. As the data exhibited in Fig. 8, TGF-β/Smad signaling pathway was notably activated in kidney of the DN model rat, and the protein expression levels of TGF-β1 and Smad2/3 were notably upregulated than those of healthy normal rat, while the Smad6/7 was downregulated. After the DN model rats received chronic treatment of silymarin, the protein expression levels of Smad2/3 and TGF-β1 or Smad7 were notably decreased or increased, respectively, in renal tissues of the DN model rat. These results strongly suggested that silymarin exhibited protective effects on the renal tissues of DN model rats may be through regulating the signaling pathway of JAK2/STAT3/SOCS1 and Smad/TGF-β, which in turn reduces the expression of downstream inflammatory factors and renal fibrosis, respectively. 4. Discussion Diabetic nephropathy is one of the major microangiopathies of T2DM, and the most critical reason for the occurrence of the end-stage renal disease [20,21]. The pathogenesis of DN is unclear, and some studies have found that the presence of persistent low-grade inflammatory response and JAK/STAT signaling pathways play critical role in occurrence and development of diabetic nephropathy [6,12,22,23]. Fallahza-deh MK et al. selected 60 patients with type 2 diabetes and massive proteinuria (urinary albumin excretion rate greater than 300 mg/24 h) who were given maximum doses of renin-angiotensin system inhibitors for more than 6 months and had an estimated glomerular filtration rate greater than 30 mL/min. Patients were randomly assigned to 2 groups, the drug treatment group and placebo group received silymarin (140 mg/day) or saline for 3 months. The results showed that the urea and creatinine decreased in the silymarin group, and the decrease was significantly lower than that in the placebo group. The urinary and serum TNF-α and MDA levels were also significantly lower in the silymarin group than those of the placebo group. In addition to the protective effect of silymarin on the nephrotoxicity model mediated by the above nephrotoxic substances, it has also been found that silymarin also alleviates iron-dextran mediated renal iron deposition in rats, protects the animal model of nephrotoxicity mediated by manganese and chemicals such as vincristine and carbon tetrachloride, and alleviates ischemia-reperfusion renal injury. In conclusion, the renal protection by silymarin is no longer limited to animal experimental studies, and clinical studies have also been gradually started. It has not only studied the protective effect of nephrotoxicity, but also studied renal interstitial fibrosis and diabetic renal disease in depth. These studies provide clinicians with new prevention and treatment ideas. So far, however, there is no study has comprehensively elucidated the mechanism of action of silymarin in improving nephropathy, including diabetic nephropathy, which affected explaining the pharmacological activity and potential toxic effects of silymarin, as well as further clinical applications. The pathogenesis of early DKD is very complex. Many evidences suggested that loss of cellular foot processes may be a strong indicator in the progression of DKD [24,25]. Podocytes are specialized cellular components in the glomerulus that are involved in constituting the glomerular filtration barrier [24]. The damage of podocytes, especially the fusion of foot processes, may lead to the development of microalbuminuria [26]. Therefore, we firstly assessed the protective effects of co-incubated silymarin on high glucose-induced damage in MPC-5 cells in vitro, and the results demonstrated the outstanding protective effects of silymarin which are exhibited in an obviously dose-dependent manner. In addition, the nephrin and podocin are important proteins of podocytes, which are important components of the hiatal membrane and play critical role in maintaining the selectivity of the glomerular filtration barrier [27,28]. Proteinuria, glomerulosclerosis and deterioration of renal function can be caused by down-regulation of nephrin and podocin proteins [29]. More importantly, nephrin and podocin can regulate the permeability of the glomerular filtration barrier [30]. If the permeability of the filtration barrier is impaired, then microalbuminuria appears. In present study, our results of in vitro cell-based assays collectively suggested that incubation of silymarin increase the cell viability of high-glucose incubated MPC-5 cell may rely on maintaining the levels of podocin and nephrin, and effectively decreased the expression of inflammatory factors. At present, the main preferred treatment for DN in clinical practice is to control blood glucose and blood pressure, but there is still no sufficiently effective improvement method for kidney injury [5]. Strict control of hyperglycemia can reduce the occurrence of DN, but hyperglycemia is not the only factor in the occurrence of DN, especially in type II diabetes, hypertension, hypertension and even obesity itself can lead to or aggravate diabetic kidney damage [31]. According to the previous literature coupled with the explanation of the etiology of DN by traditional Chinese medicine, we found that silymarin may have some application potential in the treatment of nephropathy as well as control blood glucose and improve glucose and lipid metabolism. Combined with previous studies by our group, we found that silymarin treatment can not only effectively control blood glucose, improve impaired glucose tolerance and diabetes-related symptoms, but also effectively improve renal function and renal pathological injury-related factors, including 24 h-uninary proteins, BUN and SCr levels. Previous studies have shown that increased collagen in the kidney, ECM accumulation and EMT of glomerular epithelial cells are the main factors of renal pathological changes, which will lead to DN eventually moving towards renal fibrosis [32]. In this experiment, after long-term administration of silymarin, renal fibrosis was significantly alleviated, glomerular volume and glycogen deposition were significantly reduced, and widening and matrix increase were obviously improved compared with DN model rats in the placebo group. The above results collectively suggested that long-term administration of silymarin can effectively improve pathological changes, such as glomerular septation, ultimately leading to nephron destruction, renal impairment, and even the development of irreversible glomerulosclerosis. Further detection results of the fibrosis-related indicators expression in renal tissues were notably increased or decreased in saline- or different doses of silymarin-treated DN model rats, and difference were all obvious (all p < 0.05). Above data indicated that chronic 8 weeks treatment of silymarin at different doses all could improve the kidney fibrosis by inhibiting the fibrosis- related factors expression in DN model rats. The balance between ROS production and endogenous anti-oxidant systems maintains the normal physiological condition. Numerous previous reports indicated that DN induced both ROS overproduction and decreased anti-oxidant enzyme activities. Therefore, in order to furtherly demonstrate the protective effects of silymarin, the changes of SOD, Catalase and GSH-Px, as critical anti-oxidases in antioxidant defense system in DN, were all researched. SOD, as one of the cytoprotective anti-oxidant enzymes which is capable of catalyzing the disproportionation reaction of superoxide radicals into hydrogen peroxide and molecular oxygen. CAT is a heme protein that suppresses hydroxyl radical generation and avoids peroxisome-mediated oxidative damage to cellular components. Moreover, the GSH-Px, as one of the selenium-containing enzymes could detoxify hydroperoxides and free the hydrogen into water by decreasing glutathione oxidation. In present research, chronic treatment of silymarin could effectively improve the altered activities of catalase, SOD as well as the GSH-Px in the kidney tissues of DN model rats, which collectively proved that silymarin held ability to inhibit oxidative stress by enhancing antioxidant enzyme systems and further to ameliorate kidney injuries. Furthermore, the expression of IL-8, IL-10, TNF-α, IL-6, TGF-β and ICAM-1 were obviously up-regulated in renal tissues of DN model rats compare to normal rats. Moreover, chronic treatment of silymarin obviously reversed increased expression levels of inflammatory factors in a clearly dose-dependent manner, indicating chronic treatment of silymarin could effectively improve the inflammatory indicators to further mediates the kidney injuries in DN model rats. Moreover, relative expression of activated JAK/STAT signal pathway related factors were notably up-regulated or down-regulated, respectively, in renal tissues of DN rats compare to those of normal rats (all p < 0.05). Significantly, the expression of p-STAT3 and p-JAK2 in three silymarin groups were all decreased, while the SOCS1 were all obviously increased (all p < 0.05). Not only that, we also previously found that TGF-β1 is an essential membrane-bound protein for TGF-β signaling pathway, and Smads mediate signaling in TGF-β signaling pathway which both play critical role in the development and progression of DN. The change of the expression levels of TGF-β/Smad signaling pathway-related factors, including Smad6/7, Smad2/3 and TGF-β1, in kidney lysates proved our speculation. The expression of TGF-β1 and Smad2/3 proteins were notably upregulated than normal ones, while Smad6/7 was significantly downregulated, indicating that the TGF-β/Smad pathway was notably activated. 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