Reagents
LTF, consisting of Radix Rhei Et Rhizome (18070049), Persicae Semen (19040204), Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow (1906075), and Hirudo (18050160) was obtained from the Sichuan New Green Pharmaceutical Technology Company (Chengdu, China). Recombinant spermine zinc insulin injections (Batch No. 21905202) and CaD capsules (Registration Certificate No. of imported drugs: H20140641) were purchased from Jiangsu Wanbang Biochemical Medicine Company (Jiangsu, China) and EBEWEPharma GmBH Nfg. KG (Unterach, Austria), respectively.
Animals
Male, 8-week old SD rats (Beijing Weitong Lihua Experimental Animal Technology Company, Beijing, China) were bred at the Institute of Radiology, Chinese Academy of Medical Sciences, Nankai District, Tianjin. All animals had free access to food and water and were housed at room temperature of 22 ± 2 °C with a relative humidity of 40–60%. The protocol was approved by the Animal Protection and Use Committee of the Institute of Radiology, Chinese Academy of Medical Sciences with approval No. IRM-DWLL-20200084.
Experimental design
Following an acclimatization period of 1 week, 25 of the 100 SD rats were randomly divided into the blank control group. Next, 100 SD rats fasted for 12 h, then STZ (45 mg/kg, 0.1 mol/L citric buffer, pH 0.5) was injected intravenously. The normal control group was injected with citric buffer. Random blood glucose was measured in blood samples collected from the tail vein on three consecutive days. Rats with blood glucose < 16.7 mmol/L were injected with STZ again and blood glucose was measured again on day 7. The rat diabetic model was considered to have been successfully established if the blood glucose was ≥ 16.7 mmol/L. All diabetic rats were randomly divided into three groups, (1) M group (n = 25), fed with a similar amount of intra-gastric administration of distilled water; (2) CaD group (n = 25), given a gavage of CaD (0.25 g/kg) and injected with insulin (1.5 IU/time/day) intramuscularly; (3) LTF group (n = 25), given a gavage of LTF (2 g/kg) and intramuscularly injected with insulin (1.5 IU/time/day). Normal control rats (N group), were subjected to intragastric administration of the same amount of distilled water. All rats were treated with drugs or distilled water once daily for 12 weeks.
The treatment lasted for 12 weeks, after which all animals were weighed and anesthetized with chloral hydrate. Stool samples were collected prior to anesthesia. The following specimens were collected: (1) blood; (2) serum (abdominal aorta blood was collected and centrifuged to separate the serum); and (3) rat retina.
LTF metabolite profiling using LC-MS
Accurately weighed LTF 10 daily prescriptions of granules, which composed of Rhei Radix Et Rhizoma 10 g, Persicae Semen 50 g, Hirudo 30 g, Notoginseng Radix Et Rhizoma powder 15 g and dissolved in 600 mL water. The sample was centrifuged and supernatant (200 μL) was taken. 20 μL of l-2-chlorophenyl alanine (0.3 mg/mL) dissolved in methanol as internal standard, and an 800 μL mixture of methanol and water (1/4, vol/vol) was added to each sample, and the tube was vortexed for 2 min. Ultrasonic extraction of all samples in the ice water bath for 30 min, and placed at − 20 °C for 2 h. Subsequently, the samples were centrifuged at 4 °C (13,000 rpm) for 10 min. The supernatants (150 μL) from each tube were filtered via 0.22 μm microfilters and transferred to LC vials for LC–MS analysis. Both electrospray ionization (ESI)-positive and ESI-negative ion modes of metabolites were analyzed using a Dionex Ultimate 3000 RS UHPLC (Thermo Fisher Scientific, Waltham, MA, USA). All ion modes were separated by an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). A detailed description of the protocol was previously published [15].
LTF metabolite profiling using GC-MS
Until the samples were centrifuged at 4 °C (13,000 rpm) for 10 min, the sample preparation method for GC–MS was the same as that for LC–MS. Nextly, the supernatants (150 μL) were put into glass vials and dried by concentration centrifugal dryer. 80 μL of 15 mg/mL methoxylamine hydrochloride pyridine solution was added to the glass vials, then the mixture was vortexed for 2 min and incubated at 37 °C for 90 min. 80 μL of BSTFA (with 1% TMCS) and 20 μL n-hexane were added into the mixture. 10 μL of C8, C9, C10, C12, C14, C16 (0.16 mg/mL) and C18, C20, C22, C24, C26 (0.08 mg/mL) were added as internal standards. The mixture was vortexed for 2 min and derivatized at 70 °C for 60 min. After the samples were removed, they were placed at room temperature for 30 min for GC–MS analysis. The LTF sample metabolites were measured and analyzed using an Agilent 7890 B GC + 5977A MSD (Agilent Technologies Inc., Santa Clara, CA, USA). DB-5 ms GC columns (30 m × 0.25 mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) were used to isolate compounds. The flow rate of helium (> 99.999%), as the carrier gas, was 1 mL/min. The detailed protocol has been previously presented [15].
Fundus photography (FP) and fundus fluorescein angiography (FFA)
Before anesthesia with 10% chloral hydrate (0.3 mL/0.1 kg), select rats were weighed. The right eye of each rat was dilated with compound tropicamide and then dripped with obucaine hydrochloride for anesthesia. Color FP was performed when the color of the right eye adjusted. Next, fluorescein sodium (60 µL) was injected into the tail vein for FFA examination. The fundus was observed using the Micro III retinal imaging system (Phoenix Research Labs, USA).
Measurement of BRB permeability
Rats BRB permeability was measured using Evans blue (EB), which was injected into the tail vein (3% EB; 45 mg/kg) and allowed to diffuse for 2 h. Next, the pleural cavity was opened and a needle was inserted into the aortic arch from the left ventricle. Citric acid buffer at 37 °C was continuously injected into rats until the livers turned white. The eyeballs were collected and the retinas were immediately extracted. The retinas were dried at 90 °C for 45 min in an oven to obtain the dry weight. Next, retinas were placed in 120 μL formamide at 70 °C for 18 h to extract the EB. Subsequently, the supernatants were obtained from retinas centrifuged for 45 min at 15,000 rpm. The absorbance of the supernatants was measured at 620 and 740 nm using a microplate reader (Meilin Hengtong, China). Finally, the concentration of EB was assessed according to the standard curve of EB in formamide. EB leakage = actual concentration × 120 µL. The final result according to this formula was calculated as ng/mg = EB leakage/dry weight of retinas.
Retinal thickness
Fresh eyeballs were preserved in 4% paraformaldehyde for 24 h and then embedded in paraffin. A detailed protocol has been previously described [15]. Finally, the slices were imaged using a light microscope (BX41 microscope; Olympus, Otsu, Japan). Retinal thickness was estimated using the obtained images and ImageJ (NIH, Bethesda, MD, USA).
Trypsin digestion test of retinas
Eyeballs were stored with 4% paraformaldehyde for 24 h, following which the retinas were extracted. Next, the retinas were placed in a pepsin solution at 37 °C for 1 h and trypsin solution at 37 °C for 3 h. Following dehydration, retinas were stained using periodic acid-Schiff (PAS) and imaged using a light microscope.
Western blot
The protein concentrations from retinas were quantified using a BCA assay kit (Cwbiotech, China). Proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were sealed with blocking buffer containing 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight on a shaker. The primary antibodies included rabbit anti-ZO-1 (1:1000, Proteintech, Chicago, IL, USA), rabbit anti-occludin (1:1000, Abcam, Cambridge, UK), rabbit anti-Claudin-5 (1:1000, Abcam), rabbit anti-VE-cadherin (1:1000, Abcam), rabbit anti-NF-KB (1:1000, Abcam), and rabbit anti-TNF-α (1:1000, Abcam). Then, the membranes were incubated with goat anti-rabbit IgG (H+L) -HRP (1:10,000) secondary antibodies (Jackson, USA) for 40 min at room temperature. Enhanced chemiluminescence (Millipore) was used to visualize bound antibodies and images were analyzed using the Gel Image system ver.4.00 (Tanon, China). GAPDH served as an internal reference for standardization.
RT-PCR
The RNA was extracted from the retina using a TRIzol kit (Invitrogen, Carlsbad, CA, USA). The total RNA was reverse transcribed using the HiFiScript first-strand cDNA synthesis kit (Cwbiotech) at 37 °C for 40 min and at 70 °C for 10 min. RT-PCR was performed using a SYBR PCR mixture and the process included pre-incubation at 95 °C for 10 min, amplification (45 cycles) at 95 °C for 10 s, 59 °C for 60 s, melting curves at 95 °C for 15 s, 72 °C for 15 s, and 95 °C for 15 s. Eventually, reaction samples were prepared by mixing 10 µL SYBR Master Mix (2×) Universal, 1.5 µL Primer F (10 μM), 1.5 μL Primer R (10 μM), 3 µL template, 0.5 µL ROX correction dye, and H2O (final volume, 20 µL). Relative gene expression was evaluated using the 2−ΔΔCT method.
16S rRNA gene sequencing
Fecal DNA was obtained using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The V3–V4 regions of the 16S rRNA gene were amplified via PCR utilizing standard primers. The primers included Illumina 5′ overhang adapter sequences for two-step amplicon library building, following the manufacturer's instructions for overhang sequences and barcodes. Barcoded PCR products were purified and quantified using a DNA gel extraction kit (Axygen, Union City, CA, USA) and an FTC-3000 TM real-time PCR system (Funglyn Shanghai, China), respectively. PCR products were blended at equal ratios. Next, dual 8-bp barcodes were used for multiplexing. After eight PCR cycles, two unique barcodes were merged at either end of amplicons. The library was purified and sequenced using the Illumina Novaseq 6000 platform and 250-bp paired-end reads by TinyGen Bio-Tech (Shanghai, China) Co., Ltd.
Raw Fastq files were demultiplexed according to the used barcodes. Low-quality base pairs were eliminated from PE reads, according to the following condition (SLIDINGWINDOW: 50:20 MINLEN: 50). Fastq sequences were combined adopting FLASH program software (version 1.2.11) with default parameters. Low-quality contigs were eliminated according to the screen.seqs command with the condition. Data were analyzed with several software, including mothur (version 1.33.3), UPARSE (usearch version v8.1.1756, http://drive5.com/uparse/), and R (version 3.6.3). Operational taxonomic units (OTUs) were obtained after reads were clustered in a 97% sequence. OTU representative sequences contrasted with certain databases, including Silva 128, following the use of mothur (classify.seqs) software. Finally, the remaining OTUs were used for later analysis, including phylum, class, order, family, genus, and species.
Measurement of glucose metabolism and serum cytokines
Glucose metabolism indicators, including HbA1c and FBG levels, were detected using a biochemical analyzer. Serum inflammatory parameters, including ICAM-1, IL-6, IL-8, MCP-1, VCAM-1, VEGF, and IL-1β were measured using ELISA kits.
Statistics
Data analyses were performed using the IBM SPSS Statistics 26 (Chicago, IL, USA). Data are expressed as mean ± standard deviation. Statistical analyses, including t-tests, one-way ANOVA, and nonparametric tests were performed as appropriate, depending on the conditions. For gut microbiota data, the Kruskal–Wallis test was used to compare between the groups. Kruskal–Wallis test was performed using the “ggpubr::compare_means” function from the “ggpubr” package in R to measure the significant changes in α diversity between different groups. Based on the Bray Curtis distance, analysis of similarity (ANOSIM), and principal coordinate analysis (PCoA), which compare between group similarities, were performed using the “vegan” and “ape” package in R. Spearman’s correlation coefficient was used to analyze the relationships between groups.