2.1. Sanziguben Polysaccharides

The medicinal materials of Sanziguben Recipe were purchased from Zhongshan Zhongzhi Traditional Chinese Medicine Co., Ltd. and identified. The voucher specimens (JYZ-20-ZJN, YGZ-20-ZJN, WWZ-20-ZJN, and JGL-20-ZJN, respectively) were deposited at the authors’ laboratory in the Guangzhou University of Chinese Medicine. The pulverized medicinal materials were degreased by refluxing with petroleum ether, and the dried medicinal powders were extracted twice in distilled water at 100°C under reflux for 2.5 hours each time, and the filtrates were combined and concentrated. We added ethanol to the concentrated solution to 80% of the concentration, stood still, and separated to obtain precipitation. Free proteins from polysaccharide solutions were removed by the Sevage method [23]. Finally, the solutions were concentrated and lyophilized to obtain SZP. Chemical composition analysis showed that the total sugar, uronic acid, sulfate, and protein contents of the obtained SZP were 53.06 ± 1.53%, 29.14 ± 0.73%, 5.60 ± 0.33%, and 7.95 ± 0.28%. The FT-IR (Nicolet™ iS™ 5N; Thermo Fisher Scientific) spectrum of SZP [24] and analysis of monosaccharide composition based on HPAEC-PAD (ICS5000; Thermo Fisher Scientific, Waltham, MA, USA) are shown in Supplementary Figure 1.

2.2. Reagents

Streptozotocin (STZ) was purchased from Sigma-Aldrich (Saint Louis, MO, USA); metformin was purchased from Sino-American Shanghai Squibb Pharmaceuticals Ltd (Shanghai, China); Super-Bradford Protein Assay Kit and BCA Protein Assay Kit were purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China); creatinine (Cre), total cholesterol (T-CHO), urea nitrogen, triglyceride (TG), malondialdehyde (MDA), catalase (CAT), and Superoxide Dismutase (SOD) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); Mouse TGF-β1 ELISA Kit was purchased from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China); E-cadherin (ab76319), ZO-1 (ab96587), NQO1 (ab80588), and HO-1 (ab13248) were purchased from Abcam (Burlingame, CA, USA); vimentin (10366-1-AP), α-SMA (14395-1-AP), and Nrf2 (16396-1-AP) were purchased from Proteintech (Wuhan, China); Smad7 (sc-365846) was purchased from Santa Cruz Biotechnology, Inc. (Delaware Avenue, Santa Cruz, California); Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China); Recombinant human TGF-β1 (HEK293 derived) was purchased from Peprotech (Cranbury, NJ, USA).

2.3. Animals and Experimental Design

Male SPF C57BL/6J mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were 7 weeks old and weighed 22 ± 2 g. All animal care and experimental studies were approved following the guidelines of the Animal Ethics Committee of Guangzhou University of Chinese Medicine (license number: SCXK 2019-0202). Ethical review no. ZYD-2021-013, review date: January 29, 2021. After one week of adaptive feeding, the mice were fasted for 4–6 hours and treated with STZ (dissolved in pH 4.2 citric acid buffer) 50 mg/kg, continuously injected for 5 days. After one week, the fasting blood glucose of mice greater than 16.7 mmol/L was determined to be a model of diabetes. We continued to feed mice for 4 weeks, during which the urine protein levels were tested. When the STZ injection group and the normal group showed significant differences in urine protein, we started a gavage procedure for the following groups: control group; model group; MET group (300 mg/kg/day metformin); SZP low-dose group (SZP-L, 250 mg/kg/day SZP); SZP high-dose group (SZP-H, 500 mg/kg/day SZP). The control group and model group were given equal proportions of drinking water. The administration was continued for 8 weeks, during which weight was monitored, the amount of food and water consumed was measured, and fasting blood glucose was measured every two weeks. The animal experimental protocol is shown in Figure 1(a). Urine was collected 24 h after the administration. After sacrificing the mice, blood was taken from the orbital vein, and the serum was obtained by centrifugation at 3,500 rpm for 10 minutes; the liver and kidney were weighed and stored in tissue fixative or −80 refrigerator.

2.4. Cell Culture

Human proximal renal tubular epithelial cells (HK-2 cell) and ShRNA constructed Nrf2 stable knockdown HK-2 cell line (HK-2-Nrf2-KD cell) (purchased from Xi’an CytoBiotech Biological Technology Co., Ltd.) were cultured in DMEM/F12 (Gibco) containing 10% FBS fetal bovine serum and 1% antibiotic-antimycotic solution. The EMT model of HK-2 cells was induced by HG (30 mM glucose) combined with TGF-β1. The SZP treatment group increased the required dose of SZP according to the model group. After 5 days of continuous cultivation under the above conditions, the total protein of each group was extracted for measurement.

2.5. Kidney Pathological Staining and Immunohistochemistry

For pathological staining of kidney tissue, tissue was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin, sliced, and stained with H&E, PAS, MASSON, and Sirius red according to the instructions.

For immunohistochemistry, paraffin sections were deparaffinized to water, and the EDTA (PH9.0) antigen retrieval solution was used for antigen retrieval; sections were incubated with 3% hydrogen peroxide in the dark for 25 minutes and washed with PBS. Then, 3% BSA was used for blocking at room temperature for 30 minutes and washed and incubated overnight at 4° with the primary antibody TGF-β, E-cadherin, or α-SMA. Sections were washed with PBS, and then the secondary antibody (HRP-conjugated) was added and incubated at room temperature for 50 min. Sections were washed and then slightly dried. DAB was added as a color developing solution, which was monitored under the microscope for optimal time. The section was then washed with tap water to stop the color development, the cell nucleus was counterstained, and the section was dehydrated and mounted.

2.6. Detection of Biochemical Indexes

All serum or tissue biochemical indicators were tested according to the instructions of the corresponding kit.

2.7. Cell CCK-8 Assay

HK-2 cells were seeded in 96-well plates at 1 × 10⁴ cells/well, cultured for 24 h to adhere, and then treated for 48 h in the following groups: 200 μL/well of the cell-free blank medium group, HK-2 cells were treated with SZP groups 200 μL/well (SZP administration concentrations were 0, 25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, and 500 μg/ml), and repeated 5 replicate wells. After 48 hours, the medium in the wells was discarded and a new DMEM/F12 complete medium and 10 μL of CCK-8 reagent were added to each well and incubated in the incubator for 1.5 hours in the dark. Finally, the OD values were detected at 450 nm of the microplate reader for statistics.

2.8. Western Blot

The kidney tissue was homogenized with RIPA lysis solution and centrifuged to obtain the total renal protein supernatant. Cells were washed twice with PBS, and then the lysis buffer was added and collected in a centrifuge tube. The tubes were centrifuged to obtain the HK-2 cell total protein supernatant. BCA kit was used to detect protein content. After adding the loading buffer, the protein was denatured at 100°C for 5 minutes, and the protein was separated by SDS-Page gel electrophoresis. After transfer to a membrane, the membrane was blocked with 5% skimmed milk powder. The membrane was incubated with a primary antibody overnight at 4°C, and then the HRP-conjugated secondary antibody was used for incubation at room temperature for 1 hour. After adding the chemiluminescence substrate, we used an automatic chemiluminescence instrument for exposure.

2.9. Statistical Analysis

All data are expressed as mean ± SD. P<0.05 was considered statistically significant. There was statistical significance using one-way ANOVA followed by LSD or Dunn’s test for multiple comparisons. GraphPad prism 8 software was used to graph data. Image J software was used for immunohistochemistry and western blot quantification.

3.1. SZP Improves the Disease Status of STZ-Induced DN Mice

During the administration period, the body weight, blood sugar, diet, and drinking were measured. In our experiment, the weight of the diabetic mice was significantly lower than that of the normal group. During the administration period, the weight of the mice in the normal group increased with time and the weight of the model mice decreased due to the influence of diabetes. After 8 weeks administration, body weight of mice-administered metformin and SZP were improved compared with the model group (Figure 1(b)). The fasting blood glucose of normal C57BL/6J mice was in the range of 5–10, compared to 20–33 in the STZ-injected mice. After administration, the blood sugar of diabetic nephropathy mice treated with metformin and SZP-H decreased significantly (Figure 1(c)). By measuring the water intake and diet of the mice weekly, it was found that the SZP treatment can improve the symptoms of polyphagia and polyphagia in diabetic mice (Supplementary Figure 2). The kidney index is shown in Figure 1(d), mice in the model group developed kidney enlargement, and SZP treatment reduced the kidney index.

3.2. Effects of SZP on Renal Function in STZ-Induced DN Mice

In our experiment, 24 h urine protein, serum creatinine, and urea nitrogen of model group mice were significantly higher than those of normal mice and the urinary protein content was 10 times higher than the normal group, suggesting that there is kidney dysfunction in mice with diabetic nephropathy. SZP significantly reduced 24 h urine protein measures, serum creatinine, and urea nitrogen levels, which indicate that SZP can improve diabetes-induced kidney damage (Figure 1(e) and 1(f)). By observing the T-CHO and TG, we found that DN mice had abnormal blood lipid metabolism. The T-CHO and TG of the model group were significantly increased, and these two indicators were also reduced after SZP treatment (Figures 1(h) and 1(i)). These indicate that SZP has protective effects on kidney function and blood lipid metabolism in diabetic nephropathy mice.

3.3. SZP Reduces Kidney Damage and Fibrosis in DN Mice

We observed the pathological changes in mouse kidney tissue, as shown in Figure 2. HE staining of the model group showed that there was glomerular mesangial hyperplasia, glomerular sclerosis, vacuolar degeneration of renal tubular epithelial cells, and compensatory renal tubule dilation (marked by arrows). SZP treatment attenuated glomerular damage and renal tubular disease. In PAS staining, the thickening of the basement membrane of the renal tubules in the model group was observed (marked by arrows), but the basement membrane of the tubules returned to normal in SZP-L and SZP-H groups. MASSON staining showed a large number of blue-stained collagen fibers in the tubular interstitium of the model group (pointed by arrows), and the area of collagen fibers decreased after metformin and SZP treatments. Similarly, Sirius red staining showed that there were a large number of red-stained collagen fibers in the tubular interstitium (pointed by arrows). Furthermore, under the polarized light microscope, the yellow strong refractive type I collagen fibers in the model group increased significantly, while the green weak refractive type III collagen fibers did not change significantly. Both SZP-L and SZP-H significantly reduced type I collagen fibers but had no significant effect on type III collagen fibers.

3.4. SZP Can Inhibit the Kidney EMT in Mice with DN

E-cadherin and ZO-1 are the proteins associated with tight junctions of epithelial cells, while α-SMA and vimentin are markers of mesenchymal cells. During the EMT process, there is the loss of tight junction proteins and elevated mesenchymal markers. Immunohistochemical analysis showed that E-cadherin in the renal tubules of model mice was decreased and α-SMA increased (Figures 3(a), 3(b), 3(c)). Similarly, western blot detection of total renal tissue protein found that the expressions of E-cadherin and ZO-1 proteins in the model group were decreased, but α-SMA and vimentin were significantly increased. This phenomenon was reversed after the administration of metformin and SZP (Figures 3(d), 3(e), 3(f), 3(g), 3(h)). This indicates that EMT occurred in the kidney of diabetic nephropathy mice, and SZP can prevent the occurrence of EMT to a certain extent. This may be why SZP reduces DN renal tissue fibrosis.

3.5. SZP Improves Oxidative Stress Levels in DN Mice by Activating the Nrf2 Signaling Pathway

In Figures 4(a), 4(b), 4(c), the expression of serum MDA in the model group was shown to increase, but it decreased significantly after SZP administration. The levels of SOD and CAT in the kidney of DN mice were significantly lower than those in the normal group, while expression in the SZP groups was significantly increased after treatment. MDA is a lipid peroxide produced during oxidation reaction, and the increase in its content indicates that the body is attacked by free radicals. Since SOD and CAT exert antioxidant effects in vivo, the content of both suggests the body’s ability to scavenge oxygen free radicals. The above results indicate that SZP administration improves the level of oxidative stress in DN. By detecting Nrf2 and its downstream markers HO-1 and NQO1 in the total protein of kidney tissue, we found that SZP-H significantly increased the level of Nrf2, HO-1, and NQO1 (Figures 4(d), 4(e), 4(f), 4(g)). Therefore, these results indicate that SZP could improve the oxidative stress level of DN mice by activating the Nrf2 signaling pathway.

3.6. SZP Inhibits the Expression of EMT-Related TGF-β1/Smad7 Signaling Pathway in Diabetic Nephropathy Mice

Through the detection of serum TGF-β1, we found that the serum TGF-β1 content of mice in the model group increased, which was significantly decreased after the administration of SZP (Figure 5(a)). Using immunohistochemistry (Figure 5(b)) and western blot analysis (Figures 5(c), 5(d), 5(e)) measured changes in TGF-β1 in the kidney tissues of mice. TGF-β1 was significantly increased in the model group, while TGF-β1 in the MET, SZP-L, and SZP-H administration groups was significantly lower than that of the model group. Furthermore, Smad7 plays an antagonistic role in the TGF-β1 signaling pathway and showed the opposite trend, and SZP increased the expression of smad7. This indicates that the effect of SZP’s inhibition of EMT is closely related to its inhibition of the renal TGF-β1 signaling pathway in DN mice.

3.7. SZP Improves EMT Development in Human Renal Tubular Epithelial Cells through Nrf2/Smad7

As shown in Figure 6(a), we observed the effect of different doses of SZP on the proliferation of HK-2 cells through the CCK-8 experiment. The results showed that the dose of SZP greater than or equal to 100 µg/ml had a promoting effect on the proliferation of HK-2 cells. In order to exclude the interference of this phenomenon on the experimental results, we chose 50 µg/ml as the treatment dose. In Figure 6(b), we observed that SZP treatment ameliorated HG plus TGF-β1-induced loss of E-cadherin and gain of vimentin in HK-2 cells. Moreover, Nrf2 expression was elevated after SZP treatment. These were consistent with the results found in mice. In addition, we observed the morphology of HK-2 cells. Compared with the normal group, HK-2 cells in the model group showed significant morphological changes, with sparse cell arrangement and fibroblast-like changes, arranged in a long spindle-shaped vortex, which were improved after SZP treatment (Supplementary Figure 3).

In addition, compared with HK-2 cell, the protein expression of Nrf2 was significantly reduced in the HK-2-Nrf2-KD stable cell line (Figure 6(c)). HG combined with TGF-β1 induced HK-2-Nrf2-KD cells to produce the same EMT changes as HK-2 cells. We observed that Nrf2 was not significantly elevated by SZP in HK-2-Nrf2-KD cells. After SZP administration, the expression levels of E-cadherin and vimentin were reversed in HK-2 cells, while the effect of SZP disappeared in HK-2-Nrf2-KD cells. Moreover, the expression of Smad7 was significantly increased in HK-2 cells after SZP treatment; however, this phenomenon decreased with the knockdown of Nrf2. Similarly, SZP treatment reduced the expression of TGF-β1 in HK-2 cells induced by HG combined with TGF-β1, and the effect of SZP was weakened in the HK-2-Nrf2-KD group (Figures 6(d) and 6(e)). Above results suggest that the effect of SZP on improving EMT is inseparable from the activation of Nrf2. In addition, the regulation of the TGF-β1/Smad7 pathway by SZP is achieved in part by increasing the expression of Nrf2.