4.1 Chemicals and reagents

Streptozotocin (STZ) was purchased from Sigma-Aldrich Company. Blood glucose meter was purchased from Sinocare Inc (Hunan, China). ELISA kits specific for glycosylated serum protein (GSP), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), blood urea nitrogen (BUN), creatinine (Cr), total superoxide dismutase (T-SOD), reactive oxygen species (ROS), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). All other chemicals and reagents used in the experiments were analytical grade.

4.2 Plant material

T. glandulifera root tubers were collected during November in the year 2019 from Wubao Mountain, Yunlong County, Dali Bai Autonomous Prefecture, Yunnan Province, P. R. China. The plant material was identified and authenticated by Prof. Baozhong Duan of College of Pharmacy, Dali University. The voucher specimen of T. glandulifera (NO. 20191126) was deposited in our lab.

4.3 Preparation of experimental samples (TGB and TGC)

The roots of T. glandulifera were washed to remove debris and then shade-dried. The sliced roots (2 kg) were extracted with 95% ethanol (1:3, w/v) at room temperature for 24 h, which repeated three times. And the liquid part of the ethanol was filtered out. The residue was extracted by distilled water at 100 ℃ for 4 h, the mixture was filtered, repeated three times. The filtrate was combined. The protein was removed by Sevage method for 3 times (polysaccharide solution: chloroform: n-butanol = 1:0.4:0.1) [29]. After removing the proteins, the polysaccharide solution underwent chromatographic elution using a macroporous resin AB-8 column. The eluent used was distilled water, and the resulting eluates were collected. The aqueous solution was concentrated and mixed with anhydrous ethanol to a final concentration of 65% ethanol, then left at 4 ℃ for 12 h. The TGC was obtained by lyophilizing the precipitate. Polysaccharides in TGB were precipitated with 90% (v/v) ethanol by adding anhydrous ethanol into the supernatant solution.

4.4 Cell culture

Human renal mesangial cells (HRMCs) were previously bought from Jennio Biotech Co., Ltd (Guangzhou, China). HRMCs were cultured in RPMI 1640 medium (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA) and 1% mixed antibiotic (penicillin–streptomycin, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37 ℃ with 5% CO2. The growth medium was replaced every 24 h.

4.5 TGB and TGC pretreatment and determination of HRMCs viability

The TGB and TGC were dissolved in RPMI 1640 medium containing 5.5 mM glucose. The solutions were then diluted with the same medium to obtain concentrations ranging from 50 to 800 μg/mL (50, 100, 200, 400 and 800 μg/mL). HRMCs in the logarithmic growth phase were dispersed with 10% FBS containing RPMI 1640 medium after a digestion with 0.25% trypsin, and then seeded at a density of 2 × 104 cells/well in a 96-well plate. The plate was incubated at 37 ℃ for 24 h in a humidified atmosphere containing 5% CO2. Extracts were then added to the corresponding wells and incubated for a further 48 h.

After incubation, the media in the wells were replaced with MTT reagent (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and incubated for 4 h. Subsequently, the supernatant was discarded, and 150 μL of DMSO was added to each well. The optical density at 490 nm was measured using the Bio-Tek synergy HT microplate reader (Gene Company Limited, Wuhan, China). Cell viability was expressed by MTT optical density.

4.6 High glucose-induced HRMCs proliferation model

After digestion and counting, HRMCs in the logarithmic growth phase were seeded at a density of 2 × 104 cells/well in a 96-well plate in RPMI 1640 (with 5.5 mM glucose) supplemented with 10% FBS and 1% double antibiotic and incubated at 37 ℃ for 24 h in a humidified atmosphere containing 5% CO2.

The normal glucose (NG) group had a concentration of 5.5 mM, while the high glucose (HG) group had 30.0 mM. The concentration of HG was determined by pre-test. The TGB and TGC groups were treated with drug interventions at the concentrations of 50, 200 and 800 μg/mL. The culture media for all groups, except for NG, maintained a glucose concentration of 30.0 mM. The extracts were added to designated wells at concentrations of 50, 200 and 800 μg/mL. The volume of culture medium in each well was subsequently supplemented with RPMI 1640 containing the specified glucose concentrations, bringing it to a total of 100 μL. The cells were then incubated for an additional 48 h. MTT assay was conducted to detect cell viability following above instructions.

4.7 Animals

Healthy male SPF BALB/c mice (weight 20 ± 2 g) were procured from Hunan Sja Laboratory Animal CO., Ltd (Changsha, China). Animal license No: SCXY (Xiang) 2019–0004. All animals were housed in individual polypropylene cages (10 mice/cage), reared under SPF condition (22–25 ℃, humidity of 50 ± 10%, 12 h light/12 h dark cycle), and ate and drank freely. The animals were acclimatized to the animal house for one week before experiments were performed. All the experimental protocols were strictly implemented in accordance with the Animal Care and Use Committee, and best efforts were made to minimize the pain of experimental animals.

4.8 Diabetes induction and treatment

The model of DM mice induced by STZ was established in accordance with methodologies outlined in prior research, albeit with slight modifications [3]. The dosage for the administration of the positive control drug (Tempol, a SOD mimetic) was determined based on these established protocols [3, 14]. After a week of acclimatization, the animals were randomly divided into normal control group (n = 10), DM model group (DM, n = 80). Each mouse in the control group received 0.1 M citrate buffer (solvent), while the mouse in the model group was intraperitoneally injected with 120 mg/kg STZ prepared in 0.1 M citrate buffer as a single dose. Three days after administration, the blood of mouse was collected from caudal vein to measure FBG. Mouse with FBG level ≥ 11.1 mmol/L was considered diabetic and were used for the study.

Then, diabetic mice (FBG level ≥ 11.1 mmol/L) were selected and randomly divided into 8 groups, including the model group, Tempol group (90 mg/kg), TGB (30, 60, 120 mg/kg) groups and TGC (30, 60, 120 mg/kg) groups, with each group comprising 10 animals. Administration doses were determined by pre-tests, and 30, 60, and 120 mg/kg doses were finally applied. Next, the animals underwent continuous intragastric gavage of drugs for 6 weeks, with the control group and the model group receiving an equivalent volume of distilled water.

In addition, body weight of mice was recorded once a week. Blood samples were collected from each group of animals to measure FBG.

4.9 Preparation of serum and kidney tissues

At the end of treatment, the mice were anaesthetized. The blood was collected from mice's eyes and centrifuged at 3000 r/min for 10 min after being left undisturbed at 25 ℃ for 60 min to obtain the serum, and stored at − 80 ℃ for further analysis. The mice were sacrificed by cervical dislocation, then the kidney tissues of the mice were quickly removed, weighed, thoroughly washed with ice-cold phosphate buffer saline (PBS, pH 7.4) and organ index was determined. The left kidney was homogenized in ice-cold PBS and stored in liquid nitrogen for further assays. The right kidney was fixed in 4% polyformaldehyde and embedded in wax for histopathological examination.

4.10 Biochemical parameters analysis

The TG, TC, HDL-C, LDL-C, GSP, MDA, T-SOD, GSH-Px, ROS, BUN and Cr levels were determined using ELISA kits. All these indicators were measured based on the manufacturer’s guidelines.

4.11 Histology assay

Fixed kidney tissues were embedded in paraffin, and cut into 5 μm thick slices for pathological analysis. After dewaxing and dehydration, the sections were stained with hematoxylin and eosin (H&E) for morphological analysis [26]. The stained tissues were observed through an optical microscope (Nicon, Japan) and images were taken for histopathological analysis.

4.12 High-performance liquid chromatography (HPLC) analysis4.12.1 Molecular weight (Mw) measurements

Gel permeation chromatography, also known as size exclusion chromatography (SEC), is used to determine the Mw of polysaccharides in TGB and TGC fractions [30]. HPLC analysis was performed by using an Agilent 1260 series HPLC system (Agilent Corp., Santa Clara, CA, USA) equipped with an Evaporative Light Scattering Detector (ELSD), and an Shodex SB-804 HQ (OHpak) column (7.5 mm × 300 mm, 10 μm, Shodex, Japan). The parameters for the ELSD were as follows: an evaporation temperature of 55 ℃, a nebulization temperature of 45 ℃, and the nitrogen flow rate of 1.3 mL/min. The mobile phase employed was ultrapure water. Flow rate: 0.5 mL/min. Column temperature: 35 ℃. Injection volume: 10 μL. The analysis samples were dissolved in water by ultrasound and filtered through 0.22 μm membrane filters before entering the HPLC system for separation.

Ten types of standard dextran, ranging from 180 Da to 2000 kDa, were used to create a control solution with a concentration of 1 mg/mL each. The samples were then dissolved into a concentration of 10 mg/mL. The standard and sample solutions were filtered and injected into the chromatographic column. The retention times (RT) were plotted against the logarithms of the Mw of the standards. Using the standard curve, the Mws of TGB and TGC were calculated using it.

4.12.2 Determination of the monosaccharide fraction of TGB and TGC

Monosaccharide composition analysis of TGB and TGC was performed by acid hydrolysis and precolumn derivatisation with 1-phenyl-3-methyl-5-pyrazolone (PMP) by HPLC as previously described [31]. Briefly, TGB (2 mg) and TGC (2 mg) were hydrolysed with 4 M trifluoroacetic acid (TFA) (1 mL) at 110 ℃ for 8 h, and the excess acid was completely removed with distilled water. The hydrolysed product and monosaccharide standards were derived using 0.5 M PMP methanol solvent and 0.3 M NaOH solution at 70 ℃ for 1 h, followed by neutralisation with 0.3 M HCl and evaporation. The derived products were detected by HPLC equipped with a DAD detector at 245 nm and Zorbax Eclipse XDB-C18 (4.6 mm × 250 mm, particle size 5 μm, Agilent Technologies, CA, USA). The mobile phase consisted of acetonitrile (A) and phosphate buffer solution (B). Elution was performed at a flow rate of 1.0 mL/min at 30 ℃ with a gradient in the range of 15:85–30:70 (A:B) from 0 to 30 min. The injection volume was 20 μL. Samples were compared to eight standard sugars, including D-glucuronic acid, D-glucosamine, D-arabinose, D-glucose, D-galactose, D-mannose, D-galacturonic acid, and L-fucose, to identify their monosaccharide composition. Regression equations and correlation coefficients were derived for standard monosaccharides by plotting the injection concentration on the x-axis and the peak area on the y-axis. The relative molar ratios of the monosaccharides were calculated from standard curves.

4.13 In-vitro antioxidant assay

DPPH Assay. A method based on that of Kao T.H. was modified [32]. Each sample solution (1 mL) was mixed with 150 μL of a methanolic DPPH solution (0.65 mM). After mixing thoroughly, the mixture was left in the dark for 30 min and the absorbance was measured at 517 nm. The positive control used the same concentration of VC solution, while distilled water was used as the blank control.

Hydroxyl radical Assay. Detection using the Fenton method [33]. The reaction mixture contained 1 mL of 6 mM ferrous sulfate solution, 1 mL of 6 mM salicylic acid solution and 1 mL of the polysaccharide solution, and the reaction was initiated by addition of 1 mL of 6 mM hydrogen peroxide. After incubation at 37 ℃ for 1 h and cooling to room temperature, the absorbance of the reaction was measured at 510 nm and the hydroxyl radical scavenging activity was calculated as described. The positive control used the same concentration of VC solution, while distilled water was used as the blank control.

Superoxide anion radical Assay. The superoxide anion scavenging activities of TGB and TGC were assessed according to the modified method of Li [33]. In brief, 1 mL of polysaccharide solution, 4.5 mL of Tris–HCl buffer (pH = 8.2, 0.05 mol/L), and 0.4 mL of 25 mM pyrogallol solution was added to initiate the reaction. After mixing thoroughly, the mixture was left at 25 ℃ for 5 min. At last, the reaction was terminated by addition of 1 mL of 8 mM HCl, and the absorbance was measured at 299 nm. The positive control used the same concentration of VC solution, while distilled water was used as the blank control.

The scavenging effect (%) was calculated as follows:

scavenging effect (%) = (Ablank-Asample)/Ablank.

4.14 Statistical analysis

All data are from over three independent repeated experiments and processed by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). The results are presented as mean ± standard deviation (SD). For the statistical analyses, one-way analysis of variance (ANOVA) was used followed by Tukey’s post hoc test, P < 0.05 was considered significantly different.