Synthesis and characterization of skeletal muscle particular LNP@miR-130a
Ginger nanoparticles had been remoted and purified as described in reference [5], after which phospholipid suspensions had been ready utilizing an ULtrasonic Cell Disruptor (with a sound energy of 500 W and frequency of 30 kHz) for 15 min at room temperature (25 °C).
LNP formulations had been ready utilizing a process just like a technique beforehand described for mRNA [65, 66]. Briefly, we dissolved three lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, and phosphatidic acid) with dichloromethane (DCM) in glass tubes and blended them in a ratio of three:2:5, which mimics the phospholipid composition combination. Then, phospholipid composition combination, LRP3, MYF5, MYOG, MYHC-2 A, and MYHC-2B had been dissolved in ethanol at molar ratios of 10:1:1:1:1. The lipid combination was mixed with 20 mM citrate buffer (pH 4.0) containing miR-130a at a ratio of 1:12 (ethanol/aqueous) utilizing a microfluidic mixer. Formulations had been diluted with 10 occasions the quantity of PBS and dialyzed in dialysis cassettes for no less than 24 h, and the combination was then concentrated utilizing Amicon ULtra centrifugal filters. The LNP formulations had been saved at 4 °C.
The nanoparticle morphology was decided through the use of transmission electron microscopy (TEM; Hitachi H-7500, Japan). The dimensions distribution and focus of LNPs had been examined by nanoparticle monitoring evaluation with ZetaView PMX 110 (Particle Metrix, Germany) and the corresponding software program ZetaView 8.04.02. LNPs had been diluted in PBS to measure the particle measurement and focus. NTA measurements had been recorded and analyzed at 11 positions. Calibration of the ZetaView system was carried out utilizing 110 nm polystyrene particles. All procedures had been carried out at room temperature. In response to earlier analysis [67], an agarose gel electrophoresis assay was carried out to judge the miRNA loading capability of LNP.
Animal and pattern assortment
To ascertain a high-fat mannequin, 60 male C57BL/6J mice (7 weeks outdated, bought from the Experimental Animal Heart of Xi’an Jiao-Tong College) had been acclimated to the atmosphere for one week and divided into 5 teams. 20 mice had been fed a daily weight-reduction plan (Management Food plan, CD, 10% fats) because the management group. The remaining mice had been fed a high-fat weight-reduction plan (Excessive-Fats Food plan, HFD, 60% fats) to induce weight problems. After 14 weeks, the high-fat mannequin was efficiently established, and drug administration commenced. Within the CD group, 10 mice had been untreated, 10 mice had been handled with LNP@miR-130a. Within the HFD group, 10 mice had been left untreated, 10 mice had been handled with bare miR-130a solely (supplied by Hanheng, China), 10 had been handled with Lipid Nanoparticles (LNP) solely, and 10 had been handled with LNP encapsulated miR-130a (LNP@miR-130a). Mice had been handled with LNP at a dosage of 10 mg/kg physique weight. The management group was handled with 0.9% NaCl. Injections had been administered seven occasions over a interval of three weeks. Dwell experiments started at 17 weeks, and mice had been euthanized at 20 weeks. Skeletal muscle, white adipose tissue, brown adipose tissue, liver, and serum had been collected for additional evaluation. The animal experiments had been authorised by the Analysis Ethics Committee of Northwest A&F College (approval quantity 2011-31101684).
Chemical imaging
To elucidate the biodistribution of lipid nanoparticles encapsulating miR-130a (LNPs@miR-130a), the IVIS Lumina Imaging System (Caliper Life Sciences, Hopkinton, MA, USA) was employed for in vivo fluorescence imaging [68]. LNPs@miR-130a coupled with the near-infrared fluorescent dye Cy7 permits visualization and monitoring of nanoparticles in mice to realize deep tissue penetration and minimal background autofluorescence.
Thermal imaging evaluation
Mouse topics had been evaluated thermographically utilizing a high-resolution infrared imaging system, FLIR ONE PRO Thermal Digital camera (FLIR Methods, Wilsonville, OR, USA) [69]. Subsequently, the captured thermograms had been analyzed intimately utilizing FLIR software program to course of and improve the infrared photographs, permitting correct temperature measurements and detection of thermal patte.
Glucose tolerance take a look at (GTT), insulin tolerance take a look at (ITT), and pyruvate tolerance take a look at (PTT)
Previous to the graduation of metabolic assays, mice had been subjected to fasting to standardize the experimental circumstances. Particularly, for the GTT and PTT, a 16-hour fasting interval was carried out, whereas for the ITT, a 4-hour fasting interval was noticed.
After the fasting interval, every mouse was weighed to acquire its baseline weight. A small part of the mouse’s tail was excised and gently pressed to induce blood move, after which blood samples had been collected for evaluation.
Fasting blood glucose ranges had been decided utilizing a calibrated blood glucose meter (YUWELL, Jiangsu, China), which gives a speedy and correct measurement of glucose concentrations within the blood samples. Following the baseline glucose measurement, a glucose resolution (1.5 g/kg physique weight), insulin (0.65 U/kg physique weight), or sodium pyruvate (1.5 g/kg physique weight) was administered by way of intraperitoneal injection.
After injection, blood glucose ranges had been monitored at particular time intervals (15, 30, 60, 90, and 120 min) to seize the dynamic response of glucose metabolism in mice after injection of the problem substance to evaluate modifications in glucose homeostasis and insulin sensitivity [70].
Working take a look at, swimming take a look at, and suspension take a look at
To guage the bodily endurance and train capability of mice, every having undergone an 18-week remedy routine, had been subjected to a collection of standardized bodily efficiency checks. The protocols seek advice from the next references and be modified in response to the precise scenario [71,72,73].
Treadmill fatigue take a look at
The treadmill fatigue take a look at was employed to quantify the endurance capability of the mice. Earlier than the graduation of the take a look at, the themes underwent a three-day acclimatization interval on an electrified treadmill, throughout which they had been required to run at a uniform velocity of 10 m per minute for a period of 30 min. The precise fatigue take a look at initiated on the similar velocity of 10 m/min, with the speed incrementally elevated by 1 m/min at every minute interval. The endpoint of exhaustion was outlined as the lack of the mouse to proceed operating after a 5-second interval of steady electrical stimulation (1 Hz, 0.15 mA, 163 V) delivered to the treadmill grid.
Swimming take a look at
The swimming capability of the mice was assessed utilizing acrylic resin cylinders with a diameter of 19 cm and a peak of fifty cm, crammed with faucet water maintained at a temperature of 23–24 °C, with a water depth of 20 cm. Every mouse was subjected to a load equal to 10% of its physique weight to simulate a pure swimming situation. The take a look at measured the period for which the mouse was capable of preserve its mouth and nostril above water constantly, which is an indicator of the mouse’s swimming stamina and bodily endurance. The swimming time was recorded from the second the mouse was positioned within the water till it didn’t preserve its head above water for a steady interval of three s, at which level the take a look at was terminated to forestall drowning.
Suspension take a look at
Within the suspension take a look at, mice had been inverted and suspended from a 20 cm × 20 cm steel mesh, with every small grid measuring 0.5 cm². The take a look at recorded the period for which the mice had been capable of keep their grip earlier than succumbing to gravity and dropping from the mesh. This take a look at gives a measure of the mice’s muscular energy and endurance underneath circumstances of bodily stress.
Magnetic resonance imaging (MRI)
Anesthesia was induced within the mice utilizing isoflurane to make sure their consolation and immobility throughout imaging. The mice had been positioned horizontally inside the MRI scanner to permit for a complete physique scan.
Anesthesia was induced within the mice utilizing isoflurane to make sure their consolation and immobility throughout imaging. The mice had been positioned horizontally inside the MRI scanner to permit for a complete physique scan. Anesthesia was induced within the mice utilizing isoflurane to make sure their consolation and immobility throughout imaging. The mice had been positioned horizontally inside the MRI scanner to permit for a complete physique scan.
The MRI protocol concerned the acquisition of 18 distinct slices protecting all the physique. The physique composition, together with liver and physique fats, was assessed utilizing this high-field energy MRI. Lipid quantification was carried out by analyzing the alerts from the liver fats or physique fats, with consultant photographs chosen from the center part that coated 8 slices [74]. The fourth slice from this center part was particularly used for the evaluation.
Hematoxylin and Eosin (H&E) staining
Repair muscle tissue with 4% paraformaldehyde for paraffin and frozen sections. After dewaxing, the paraffin sections had been stained with hematoxylin and eosin and noticed underneath a microscope.
Manually circle the cross-sectional form of muscle fibers and use Picture J software program (NIH) to calculate the cross-sectional space measurement. Every muscle makes use of no less than 500 fibers to calculate its cross-sectional space [79].
Fluorescence staining
The handled cells had been discarded from the tradition medium, washed with PBS for two occasions, added applicable quantity of fixative, and glued at room temperature for 30 min. Discarded the fixative, washed with PBS for two occasions, added applicable quantity of 0.5% Triton X-100, and permeabilized at room temperature for 30 min. Washed with PBS for two occasions, and closed at room temperature for 30 min with 5% BSA. Diluted the first antibody in response to the suitable proportion, and left at 4℃ in a single day. Washed with PBS for two occasions, and diluted the secondary antibody in response to the suitable proportion, and photographed after 1 h at room temperature. Dilute the secondary antibody in response to the suitable proportion, and take photos after 1 h at room temperature [75]. The identical experimental protocol was used for mice tissue part.
Succinate dehydrogenase (SDH) staining
The experimental protocol relies on the directions of the reagent equipment (Solarbio, G2000, China). Take contemporary tissue that can not be fastened, put together frozen slices, add NBT incubation resolution dropwise to the slices, and place them in a 37 ℃ incubator for five–30 min of fixed temperature staining. Gently rinse with distilled water to take away extra dye. Observe and take photographs underneath a microscope.
Myokines detection
Add 50ul of diluted customary and 50ul of take a look at pattern to every response nicely. Instantly add 50ul of biotin-labeled antibody. Cowl the plate and blend gently, incubate at 37℃ for 1 h. Discard the liquid and wash 3 occasions with washing resolution. Add 80ul of streptavidin-HRP to every nicely, combine gently, and incubate at 37℃ for 30 min. Discard the liquid and wash 3 occasions with washing resolution. Add 50ul of the corresponding substrate to every nicely, combine gently, and incubate at 37℃ for 10 min, avoiding gentle. Take away the microplate and rapidly add 50ul of cease resolution. After including the cease resolution, measure the outcomes instantly [76]. Measure the OD worth of every nicely on the specified wavelength.
Serum biochemical profiling
Following the experimental protocol, blood samples had been collected from the themes on the conclusion of the examine [77]. The samples had been centrifuged at 3000 rpm for 10 min at 4 °C to isolate the serum. The biochemical evaluation was carried out on the Biochemical Evaluation Laboratory inside the Faculty of Animal Science and Know-how at Northwest A&F College, Yangling, Shaanxi, China. A spread of biochemical parameters had been measured, together with glucose, lipids, liver operate markers, and electrolytes, utilizing validated analytical strategies and high-precision devices to evaluate the metabolic standing of the themes.
Measurement of triglyceride (TG) ranges in muscle tissue
To quantify triglyceride (TG) content material inside muscle tissue, a TG assay equipment from Solabio (Beijing, China) was employed. The absorbance was measured at 450 nm utilizing an ELISA spectrophotometer (mannequin 51119080, Thermo Fisher, Waltham, USA), which was calibrated prior to make use of to make sure correct readings [78]. TG ranges had been decided by evaluating the absorbance values to a regular curve generated from the equipment’s TG requirements.
Cell tradition and transfection
C2C12 cells (ATCC) had been cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2. Cells had been seeded into 6, 12, and 24-well plates and transfected with miR-130a mimics or inhibitors (Ribobio) at a focus of fifty nmol/L, utilizing X-tremeGene siRNA transfection reagent (Roche) and Opti-MEM medium (Gibco). After 12 h, the medium was changed with contemporary tradition medium.
To induce cell differentiation, DMEM containing 2% horse serum (Gibco) was used [79]. After 7 d of myotube formation, cells had been incubated with DMEM containing 0.5% fatty acid-free BSA and 500 μm oleic acid ester (Aladdin) for twenty-four h earlier than harvesting.
Complete RNA extraction, RNA Reverse Transcription, and RT qPCR
Tissue or cell lysate was transferred to a 1.5mL RNase-free EP tube, add an applicable quantity of TRIzol, and let it stand for five min. Add 200µL of chloroform to every tube, blended completely, and incubated on ice for 10 min and the samples had been centrifuged at 4 °C for 15 min. After centrifugation, transferred the higher aqueous section (roughly 500µL) to a brand new EP tube. The pattern was incubated on ice and precipitated with alcohol for 10 min. Centrifuge and take away the supernatant and wash the RNA pellet with 1mL of 75% ethanol. The supernatant was eliminated, and the RNA pellet was air-dried or vacuum-dried for five–10 min after which resuspended with applicable quantity of water.
The extracted RNA was reverse transcribed utilizing a two-step technique. Quantitative RT-PCR was carried out utilizing qPCR SYBR Inexperienced Premix on a real-time PCR system. The expression of the goal gene was normalized to the typical expression of the management group [80].
Luciferase reporter gene assay methodology
The plasmid pis-CHECK2, which incorporates the wild-type and mutant 3’-untranslated areas (3’-UTRs) of the PPARG gene, was sourced from Normal Biosystems Co., Ltd. (Anhui, China). HEK293T and C2C12 cells had been seeded in 48-well plates and co-transfected with the three’-UTR constructs and miR-130a mimic utilizing Lipofectamine 2000. Luciferase exercise was assessed in cell lysates using the Twin Glo Luciferase Assay System from Promega (Madison, WI) [81].
Statistical evaluation
Statistical evaluation was carried out utilizing two-way ANOVA and a number of t-test in GraphPad Prism model 8 statistical software program (La Jolla). The info offered as ± SEM imply, with P < 0.05 is taken into account important * P < 0.05, ** P < 0.01, *** P < 0.001.
