2.1 Reagents, animals, and assay kits
Specific pathogen-free (SPF) male Sprague–Dawley (SD) rats weighing 300±20 g were provided by the Animal Center of Fujian University of Traditional Chinese Medicine (FJTCM) [Animal license number: SCXK (Zhe) 2019–0002]. They were housed in an SPF laboratory with an ambient temperature of 25 °C and a 12 h light–dark cycle, and the animals had free access to standard rat diet and water. All experimental protocols were approved by the FJTCM Laboratory Animal Welfare Ethics Committee. The rats were handled in strict compliance with ARRIVE guidelines.
Cortex Lycii was purchased from the Third Affiliated Hospital of FJTCM and authenticated by Professor Che Su-rong from FJTCM. Voucher specimens were stored in the Key Laboratory of TCM in Fujian Higher Education Institutions. In this study, the following primary antibodies were used: anti-AQP5 (ab78486, Abcam), anti-M3R (ab87199, Abcam), anti-β2AR (ab182136, Abcam), anti-PRKAR1A+PRKAR1B (ab139695, Abcam), anti-calmodulin 1/2/3 (ab45689, Abcam), anti-NHERF-1 (ab3452, Abcam), anti-Raf1 (ab173539, Abcam), and anti-MEK1+MEK2 (ab178876, Abcam).
2.2 Preparation of Cortex Lycii extract
Cortex Lycii extracts were prepared through the following steps: crude Cortex Lycii powder was soaked in 75% ethanol (1:10, w/v) for 1 h, extracted at reflux for 1 h, and filtered. The residue was extracted following the same method twice, and the filtrate of the three extracts was collected and concentrated by rotary evaporation. Subsequently, 0.5% CMC-Na solution was used to dissolve the extract, and the final concentration was adjusted to 6 g/mL (clinically equivalent dose). The extract was stored at 4 °C.
2.3 UPLC/MS analysis for the quality control of Cortex Lycii extract
Cortex Lycii extract quality was monitored using an ultrahigh-performance liquid chromatography (UPLC) system (Vanquish, Thermo Fisher Scientific). The analytical conditions were as follows: Waters ACQUITY UPLC H-Class instrument and Waters UPLC BEH C18 column (1.7 m, 2.1×100 mm). The mobile phase comprised 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multistep linear elution gradient program was as follows: 0–3.5 min, 95%–85% A; 3.5–6 min, 85%–70% A; 6–6.5 min, 70%–70% A; 6.5–12 min, 70%–30% A; 12.5–18 min, 30%–0% A; 25–26 min, 0%–95% A; 26–30 min, 95%–95% A; and 26–30 min, 95%–95% A. The column temperature was set at 40 °C, the flow rate was set at 0.4 mL/min, and the sample injection volume was set at 5 L. Cortex Lycii extract was redissolved in 80% methanol18.
The MS and MS/MS data were obtained using an Orbitrap Exploris 120 mass spectrometer and Xcalibur software in the IDA acquisition mode. The mass range was 100–1500 during each acquisition cycle. From each cycle, the top four were selected and screened, resulting in the retrieval of corresponding MS/MS data. The other conditions were as follows: sheath gas flow rate: 30 Arb; auxiliary gas flow rate: 10 Arb; ion transfer tube temperature: 350 °C; vaporizer temperature: 350 °C, full MS resolution: 60,000; MS/MS resolution: 15,000. In the NCE mode, the collision energy was 16/32/48 at 5.5 kV (positive) or −4 kV (negative) spray voltage.
2.4 Experiments in animals and sample collection
After 1 week of accommodation, 36 SD rats were randomly divided into these three groups: control group (saline group, N=12), model group (YDH group, N=12), and Cortex Lycii group (DI group, N=12). The rats from the model and DI groups were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for 1 week8. Those in the control group were given an equal volume of sterile saline solution (1 mL/100 g) by gavage.
On day 8, the rats from the DI group were treated with Cortex Lycii (6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups. Body weight, body temperature, and sweat spots were measured once a day throughout the experiment. The blood sample was collected by the abdominal aortic method 1 h after the last medication, and animals were then subject to euthanasia. The blood sample was centrifuged at 3000×g for 10 min to separate the serum. The serum was aliquoted and stored at −80 °C for subsequent ELISA and proteomic experiments. The paw pads were collected from each group of rats, and the protein expression in these paw pads was determined by Western blot.
2.5 Measurement of sweat spots
Sweat coloring experiments were performed on each group of rats by using Hotan Takagaki reagent A solution (2% iodine dissolved in absolute ethanol) and B solution (soluble starch mixed with castor oil). After the sweat spots reacted with the reagent and turned blue-black, photographs were obtained. The proportion of the blue-black part of the foot pad was calculated with software image J.
2.6 ELISA
Serum samples were collected as mentioned above. Serum levels of cAMP and cGMP were measured using a commercially available ELISA kit (Commercially available) following the manufacturer’s instructions19.
2.7 TMT-LC-MS/MS
The serum sample from the animal experiment was used in proteomics analysis. PierceTM Top 14 Abundant Protein Depletion Spin Columns (ThermoFisher Scientific) were used to remove the top 14 high-abundance proteins. Finally, the protein concentration was measured according to the manufacturer’s instructions using a BCA kit. The protein solution was reduced for 30 min at 56 °C with 5 mM dithiothreitol and alkylated for 15 min at room temperature in darkness with 11 mM iodoacetamide. Thereafter, the protein sample was diluted with 100 mM TEAB in less than 2 M urea. Trypsin was added at a trypsin-to-protein mass ratio of 1:50 for the first overnight digestion and a trypsin-to-protein mass ratio of 1:100 for the second digestion for 4 h. Finally, the C18 SPE column desalted the peptides20.
Subsequently, 0.5 M TEAB was used to dissolve the tryptic peptides. Each peptide channel was labeled with its own TMT reagent (according to the manufacturer’s procedure, ThermoFisher Scientific) and incubated at room temperature for 2 h. About 5 L of each sample was pooled, desalted, and analyzed by MS to assess the labeling efficiency. The pooled samples were quenched with 5% hydroxylamine, desalted using a Strata X C18 SPE column (Phenomenex), and vacuum centrifuged for drying. The peptides were exposed to sodium/iodide symporter sources. Tandem mass spectrometry (MS/MS) was carried out with the use of Q ExactiveTM Plus (Thermos) equipment that was linked online to a UPLC system21.
The electrospray voltage was set at 2.0 kV, the entire MS scan resolution was set to 60,000, and the scan range was 350–1600 m/z. The top 20 most abundant precursors were selected for further MS/MS analyses under a 30 s dynamic exclusion period. Normalized collision energy (NCE) of 28% was used for HCD fragmentation. The Orbitrap detected the fragments with a resolution of 30,000. The initial fixed mass was fixed at 100 m/z. The AGC target was set to 1E5, with an intensity threshold of 3.3E4 and a maximum injection time of 60 ms. The generated MS/MS data were searched against the human SwissProt database (20422 items) concatenated with the reverse decoy database using the MaxQuant search engine (v.1.6.15.0). Trypsin/P was identified as a cleavage enzyme with the capability to catalyze up to two missed cleavages. The initial search utilized a mass tolerance of 20 ppm for precursor ions, whereas the main search used a mass tolerance of 5 ppm. By contrast, the mass tolerance of 0.02 Da was used for fragment ions. Carbamidomethylation of Cys was designated as a fixed modification, whereas acetylation of the protein’s N-terminus and oxidation of Met were designated as variable modifications. FDR decreased to 1%.
2.8 Bioinformatics analysis
Molecular functions, cellular components, and biological processes of the identified proteins were analyzed using the GO database (http://geneontology). Protein signaling pathways were annotated by using the KEGG pathway database (http://www.genome.jp/kegg/ map-per.ht). Protein interaction networks were analyzed by STRING software (http://string-db.org/). Functional annotation allowed for the inference of the biological functions associated with the output protein in the context of sweating15.
2.9 Western blot
Protein was extracted from rats’ foot pads and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analyses. In brief, an appropriate amount of protein was added to each lane of 10% SDS-PAGE gel. The membranes were blocked for 15 min by using QuickBlock™ Blocking Buffer for Western blot. The blots were then incubated overnight at 4 °C with the following primary antibodies: GAPDH, β-actin, M3R, β2AR, AQP5, PKA, CaM, raf1, and NHE-RF1. At room temperature, the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The indicator proteins on membranes were observed using the BeyoECL Star chemiluminescence kit (Beyotime Biotechnology, Shanghai, China).
2.10 Effects of Cortex Lycii on isoproterenol- or pilocarpine-induced sweat secretion in YDH rats
Isoproterenol (β2-receptor agonist) or pilocarpine (M3-receptor agonist) was used to induce sweats in YDH rats to examine the effects of Cortex Lycii. A total of 36 YDH rats were randomized into six groups (each group comprised six rats): control (Group A), model (Group B), model + pilocarpine (Group C), model + pilocarpine +DI (Group D), model + isoprenaline (Group E), and model + isoprenaline + DI (Group F). The rats in groups B–F were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for 7 days, whereas the control group was given an equal volume of sterile saline solution (1 mL/100 g) by gavage.
On day 8, the rats in groups D and F were treated with Cortex Lycii (6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups. On day 20, the rats in groups C and D were administered with pilocarpine (0.035 g/kg) via subcutaneous injection. Meanwhile, the rats in groups E and F were administered with isoprenaline (0.255 mg/kg) via subcutaneous injection. Sweat spots were measured daily throughout the experiment.
2.11 Statistical analysis
All data were presented as the mean ± standard deviation (SD). The three groups were analyzed by ANOVA and post-hoc Tukey test in SPSS 21.0 software (SPSS, Armonk, NY, USA). Graphs were generated with origin 2019b software.