Drugs, reagents, and antibodies
Zishen Jiangtang Pills (ZJP) was prepared by the Pharmacy Department of Shenzhen TCM Hospital. The main components were Astragalus membranaceus (Astragalus Linn.), Codonopsis pilosula (Codonopsis Wall.), Carapax Trionycis (Trionychidae), Panax Notoginseng (Panax L.), Rehmannia glutinosa (Rehmannia), Schisandra chinensis (Schisandra Michx.), Polygonatum (Liliaceae), Achyranthes bidentata (Achyranthes L), Ostrea gigas tnunb (Ostrea), Rhizoma Drynariae (Davalliaceae) (Batch No: Guangdong Z20070085). This mixture was dissolved with warm distilled water and concentrated to a crude drug containing 1 g/mL.
The main reagents and antibodies used are as follows: Streptozocin (STZ) and carboxymethyl cellulose sodium (CMC) (No. S0130/C4888, Sigma, USA). Insulin primary antibody (No.3014s, CST, USA), fluorescent secondary antibody (No.111-585-003, Jackson Immuno Research Laboratories, inc, USA; No. A23210, Abbkine, USA), glucagon primary antibody (No.ab10988, Abcam, UK), Dapi fluorescent seal tablets (No.0100 − 20, Southern Biotech), chromatographically pure acetonitrile (Merck, USA), and the reference products including notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, icariin, and ginsenoside Rb1 (China Food and Drug Testing Institute).
A total of 70 2-month-old SPF male Wistar rats, weighing from 160 to 190 g, were purchased from the experimental animal center of Southern Medical University. The animals were housed in the SPF room in Shenzhen Municipal Center for Disease Control and Prevention, with an ambient temperature of 18–22℃, natural circadian rhythm illumination (12h: 12 h), and an environmental humidity of 40–70%. All animal experiments were in accordance with the Animal Research Ethics Committee and approved by the Animal Protection and Use Committee of the Guangdong Experimental Animal Center.
Establishment of a rat diabetes model
Rats were adaptively fed for 2 weeks, and 10 rats were randomly selected as normal controls. The remaining 60 rats were used to establish the diabetes model. After fasting for 12 h, rats were injected intraperitoneally with 0.2% STZ (diluent 0.1 M citrate-sodium buffer, pH 4.5) at a dose of 60 mg/kg. The blood glucose of the rats was measured using a blood glucose meter 72 h after administration. Rats with a fasting blood glucose > 16.7 mmol/L were fed commonly for 2 weeks. Those rats that still had a fasting blood glucose > 16.7 mmol/L were considered as successful models. The normal control group was injected intraperitoneally with the same amount of 0.1 M citrate-sodium buffer (pH = 4.5). Some rats were failed or dead in the establishment of diabetes model. Finally, a total of four groups with 10 rats in each group were obtained. The rats of four groups then received a 3-month treatment. The normal group and model group were given 0.5% sodium carboxymethyl cellulose 10 mL/kg/day via intragastric administration, and the high (3.0 g/kg/d ZJP) and low dose (1.5 g/kg/d ZJP) groups were simultaneously given with 0.5% sodium carboxymethyl cellulose 10 mL/kg/day via intragastric administration. At the end of the treatment, all of the rats were fasted for 12 h and then anesthetized using sodium pentobarbital (50 mg/kg) and sacrificed via abdominal aorta bleeding. The blood was obtained, and the pancreas and bone tissues were stored at -80℃. All animal experiments were in accordance with the Animal Research Ethics Committee and approved by the Animal Protection and Use Committee of the Guangdong Experimental Animal Center.
Hematoxylin and eosin (H&E) staining
Pancreatic tissues were sliced, fixed in 4% paraformaldehyde, rinsed with water for 24 h, dehydrated with an ethanol gradient, placed in anhydrous acetone, and embedded in polymethyl methacrylate. H&E staining was performed as follows: xylene for 5 min, xylene for 3 min, 100% alcohol for 30 s, 100% alcohol for 30 s, 95% alcohol for 30 s, 90% alcohol for 30 s, hematoxylin staining for 10–15 min, 1% hydrochloric acid alcohol differentiated slice for 10 s, 1% eosin staining for 3 min, 90% alcohol for 30 s, 95% alcohol for 30 s (2x), 100% alcohol for 30 s (3x), carbonic acid xylene for 30 s, xylene for 30 s (3x), and a neutral gum seal.
Double-label pancreatic immunofluorescence
The paraffin slices were baked at 60℃, dewaxed with xylene and ethanol, and repaired using citrate antigen repair solution. The slices were then incubated with insulin (1:400) and glucagon (1:1000) primary antibodies in the dark at 4℃ overnight. The slices were then incubated with a fluorescent secondary antibody (1:100) at 37℃ in the dark for 1 h, stained with DAPI, and sealed using a fluorescent sealing tablet. The positive islet beta cells appeared red, the positive islet alpha cells appeared green, and the nuclei appeared blue.
Pancreatic transmission electron microscopy
Pancreatic tissues were fixed with 2.5% glutaraldehyde, dehydrated in ethanol, penetrated with propylene oxide, embedded with a 1:1 propylene oxide and resin mixture for 2 h, pure resin for 2 h, and resin at 48℃ for 10 h. The samples were then sliced at 500–1000 nm, stained with toluidine blue, sliced at 70 nm, and stained with lead and uranium.
Femoral electron microscope scanning
The fixed bone tissue sections were processed as follows: sections were stained with the Weigert's iron lignin on a glass slide for 40 min and rinsed with water until it turned blue. These were then incubated in 1% hydrochloric acid ethanol and washed with water until it turned blue, dyed with Van Gieson picric acid-magenta solution for 3 min, dehydrated with 95% ethanol and anhydrous ethanol, quickly dipped in fresh anhydrous ethanol, and then blotted dry. After the above treatment, the sections were immersed in an acetonitrile solution, which was then replaced with 70%, 80%, 90%, 95%, 100% acetonitrile, soaked for 15–20 min each time, and finally replaced with 100% acetonitrile and dried. The sections were then sprayed with carbon and gold and observed under an electron microscope.
The proteins were extracted from the rats bone tissues in different groups. The extracted protein samples were subjected to reductive alkylation treatment to open the disulfide bonds for subsequent enzymatic hydrolysis of the proteins. Trypsin and 8-plex iTRAQ reagent (AB Sciex, Cat. No.4381664) were used to label the protein. The mixed peptides were pre-isolated using high pH reverse phase chromatography (High pH-RP Chromatography), and liquid chromatography was performed coupled with tandem mass spectrometry (LC-MS/MS) analysis. The mass spectrometry data was assessed using Protein Pilot software (AB, Version 5.0) and aligned for identification; the database used was UniProtKB/Swiss-Prot. The identification criteria for the differentially expressed proteins was a fold difference of ≥ 1.5 or ≤ 0.667, and the number of unique peptides per protein ≥ 2 and < 0.05 was considered to be a significant difference.
The Omicsbean(http://www.omicsbean.cn/) multi-functional bioinformatics analysis tool, integrated STRING (http://www.string-db.org) biological database, and Cytoscape software were used to perform enrichment analysis on the identified differentially expressed proteins based on GO biological process, cellular component, and molecular function. Additionally, KEGG (http://www.kegg.jp/kegg/pathway.html) biological pathway enrichment analysis was performed on the differentially expressed proteins.
HPLC fingerprinting and active ingredient detection
A reference solution that contained notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, icariin, and ginsenoside Rb1 was mixed with methanol at a concentration of 0.1 mg/mL. The ZJP powder was dissolved in 100% methanol and filtered as test solution. The reference solution and the test solution were tested via HPLC, and the characteristic maps of both solutions were obtained. The chromatographic conditions were as follows: octadecyl silane-bonded silica gel was used as filler, and a gradient elution was carried out at a detection wavelength of 203 nm. The column was an Agilent TC-C18 (250 mm × 4.6 mm, 5 µm) with water as mobile phase A and acetonitrile as mobile phase B. Gradient elution: 0–12 min, 81% A, 12–60 min, 81%-64% A, flow rate 1.0 mL/min, and a detection wavelength of 203 nm.
The obtained data were statistically analyzed and processed using State12.0 software, and the measured data were expressed as the mean ± standard deviation (± SD).One-way analysis of variance (ANOVA) was used to assess the differences between the groups, and the Bonferroni test was used for multiple comparisons between the groups. Wilcoxon rank sum test was used for comparison between the group data that did not conform to a normal distribution or variance. Statistical significance was defined as P < 0.05.