Liver-target nanotechnology facilitates berberine to ameliorate cardio-metabolic diseases

Abstract

Cardiovascular and metabolic disease (CMD) remains a main cause of premature death worldwide. Berberine (BBR), a lipid-lowering botanic compound with diversified potency against metabolic disorders, is a promising candidate for ameliorating CMD. The liver is the target of BBR so that liver-site accumulation could be important for fulfilling its therapeutic effect. In this study a rational designed micelle (CTA-Mic) consisting of α-tocopheryl hydrophobic core and on-site detachable polyethylene glycol-thiol shell is developed for effective liver deposition of BBR. The bio-distribution analysis proves that the accumulation of BBR in liver is increased by 248.8% assisted by micelles. Up-regulation of a range of energy-related genes is detectable in the HepG2 cells and in vivo. In the high fat diet-fed mice, BBR-CTA-Mic intervention remarkably improves metabolic profiles and reduces the formation of aortic arch plaque. Our results provide proof-of-concept for a liver-targeting strategy to ameliorate CMD using natural medicines facilitated by Nano-technology.

Fig. 1

Characterization of TPGTSA and BBR-CTA-Mic. a The physical properties of TPGTSA. Left: Nuclear magnetic resonance hydrogen spectrum (¹H-NMR); middle: Flourier transformation infrared spectroscopy (FTIR); right: matrix-assisted laser desorption/ionization time of mass spectrometry (MALDI-TOF-MS). b Particle size, Zeta potential and morphology of empty CTA-Mics (up) and BBR-CTA-Mics (down). c Drug release study. The release of BBR from BBR-CTA-Mic and BBR-TPGS-Mic was carried out by dialysis method. BBR-S was used as control. 0.4 mL of BBR-S or BBR containing Mics was sealed in dialysis bags and submerged in 50 mL of fasting state simulated gastric fluid (SGF, pH 1.6), fasting state simulated intestinal fluid (SIF, 5 mM of bile salts, pH 6.8), simulated serum (SS, 20% FBS albumin, pH 7.4) and simulated liver environment (20% mice liver homogenate), respectively. Data are presented as mean ± SEM (n = 5). d Trans-epithelial transport study of BBR formulations in Caco-2 cell lines. Up: bidirectional apparent trans-epithelial permeability (Papp) of BBR contained formulations and the effect of CsA on the transport of different samples; down: the effect of different BBR containing formulations on Rho123 transportation. Data are presented as mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001, vs untreated control, p-values were calculated by unpaired two-sided Student’s t-test. Scale bars, 100 nm (b)

Fig. 2

Intracellular-uptake analysis. a. The cells were treated with various BBR formulations at an equivalent BBR concentration of 1 μg mL⁻¹ for 3 h, respectively, at 37 °C in 5% CO₂. Left: representative fluorescent images of BBR in HepG2 cells visualized using CLSM (Carl Zeiss, Germany); right: representative flow cytometry diagram of BBR in HepG2 cells. b The cells were treated with BBR-S or BBR-CTA-Mic for 1, 4, or 8 h, respectively. Representative fluorescent images of BBR in HepG2 cells visualized using CLSM. c HepG2 cells were pre-incubated with BBR-S or BBR-CTA-Mic accompanied with P-gp siRNA or mismatch siRNA (mm RNA) (50 nM) for 8 h. Cells were washed with PBS twice and incubated with fresh medium for another 4 h. Left: representative fluorescent images of BBR and P-gp in HepG2 cells visualized using C2t Nikon fluorescent microscope (Morrell, USA); middle: representative flow cytometry diagram of BBR deposition in HepG2 cells before and after dispel experiment. Right: representative flow cytometry diagram of P-gp expression in HepG2 cells before and after RNAi. The experiments were conducted in five times. Scale bars, 10 μm (a–c)

Fig. 3

Bio-distribution Evaluation. C57BL/6J mice were administered with BBR-S or BBR-CTA-Mic (50 mg kg⁻¹ of BBR) via gavage injection. a At each predetermined time point, a group of five mice for each formulation were euthanized and blood (0.5 mL) were obtained from posterior orbital venous plexus to a heparinized tube and major organs (heart, liver, spleen, lung, and kidney) were harvested. Major organs were imaged using the IVIS imaging system at excitation/emissio n = 465/540 nm. (Heart, liver, spleen, lung, and kidney, from left to right). b Quantitative analysis of the distribution of BBR in C57BL/6J mice at different time points achieved via LC/MS/MS. (n = 5, mean ± SEM). Liver-focused evaluation was applied to further assay the hepatic cell accumulation of BBR in BBR-S or BBR-CTA-Mic group. The cryostat sections of liver sample were prepared by cutting the liver tissue into 4 μm. c Liver cell accumulation of BBR was visualized using CLSM LSM710 (Carl Zeiss, Germany) Ex = 365 nm; Em = 480 nm. d Liver accumulation of BBR was tested using ambient mass spectrometry imaging method. The experiments were performed on an air-flow-assisted desorption electrospray ionization (AFADESI)-MSI platform equipped with a Q-Orbitrap mass spectrometer. Scale bars, 50 μm (c)

Fig. 4

In vitro pharmacological effect. The HepG2 cells were treated with various BBR formulations at an equivalent BBR concentration of 1 μg mL⁻¹ for 8 h at 37 °C in 5% CO₂. a The induction of p-AMPK (pink), InsR (red) and LDLR (green) protein expression by different BBR formulations was probed simultaneously with anti-InsR, LDLR and p-AMPK antibodies and visualized with C2t Nikon fluorescent microscope (Morrell, USA). b The mRNA expression of AMPK, InsR, AKT, and LDLR genes was evaluated by RT-PCR. The results were normalized to GAPDH. c The protein expression of AMPK, p-AMPK, InsR, p-InsR, AKT, p-AKT, and LDLR was tested using western blot analysis. The results were normalized to GAPDH as density ratio. Data are presented as mean ± SEM (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001, vs NC group, p-values were calculated by unpaired two-sided Student’s t-test. Scale bars, 100 μm (a)

Fig. 5

In vivo pharmacodynamics. C57BL/6J mice were feed with HFD for 8 weeks, followed with randomly allocated into four groups: model control group (MC), BBR-S group (BS, 50 mg kg⁻¹ of BBR), empty micelle group (EM, same as BBR micelles) or BBR-CTA-Mic group (BM, 50 mg kg⁻¹ of BBR), accompanied with HFD for another 8 weeks by gavage. Untreated mice fed with standard rodent diet (NC) were used as control. a Representative photographs of p-AMPK (pink), InsR (red) and LDLR (green) protein expression in liver tissue of different group mice were visualized using C2t Nikon fluorescent microscope (Morrell, USA) by probing with anti-InsR, LDLR and p-AMPK antibodies simultaneously. b The mRNA expression of AMPK, InsR, AKT, and LDLR genes was evaluated by RT-PCR. The results were normalized to GAPDH. c The protein expression of AMPK, p-AMPK, InsR, p-InsR, AKT, p-AKT, and LDLR was tested using western blot analysis. The results were normalized to GAPDH as density ratio. Data are presented as mean ± SEM (n = 10), *p < 0.05, **p < 0.01, ***p < 0.001, vs mice in MC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs mice in EM group, p-values were calculated by unpaired two-sided Student’s t-test. Scale bars, 200 μm (a)

Fig. 6

In vivo hyperlipidemia, body weight and adiposity analyses. HFD-fed C57BL/6J mice were treated with various BBR formulations by gavage. Untreated mice fed with HFD (MC group) and standard chow food (NC group) were used as control. a Biochemical analyses. Plasma TG, cholesterol, LDL-c, glucose, and hepatic TG were measured with enzymatic methods using an automatic biochemical analyzer. b Representative pictures and weight changes of whole body, mesentery and epididymal fat, liver tissue and size change of adipocyte. Data are presented as mean ± SEM (n = 10), *p < 0.05, **p < 0.01, ***p < 0.001, vs mice in MC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs mice in EM group, p-values were calculated by unpaired two-sided Student’s t-test. Scale bars, 100 μm (b)

Fig. 7

Fatty liver and hepatic injury evaluation. HFD-fed C57BL/6J mice were treated with various BBR formulations by gavage (n = 10 for each group). Untreated mice fed with HFD or standard chow food (NC group) were used as control. a Representative photographs of HE-stained liver sections. Insets contain images of whole liver tissue section. The regions of interest (ROI) are boxed in white, and their magnified images are shown at the right. b Representative photographs of oil Red O stained liver sections. The regions of interest (ROI) are boxed in white, and their magnified images are shown at the right

Fig. 8

Inflammation condition analysis. HFD-fed C57BL/6J mice treated with various BBR formulations by gavage. a Pro-inflammation cytokine levels in plasma. Following the termination of the experiment, blood samples were collected and used for the determination of plasma TNF-α, IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, MCP-1, MMP-9, and IFNγ levels by enzyme linked immunosorbent assay (ELISA) according to instruction of the manufacturer with a illuminometer at 490 nm. The tissue of epididymal fat and liver were harvested. b Representative photograph of immunofluorescent stained liver tissues for IL-6 (red) and TNF-α (green) visualized using C2t Nikon fluorescent microscope (Morrell, USA). The regions of interest (ROI) are boxed in white, and their magnified images are shown at the bottom. c Representative photographs of immunofluorescent stained adipose tissues for IL-6 (red) and TNF-a (green) visualized using C2t Nikon fluorescent microscope (Morrell, USA). The regions of interest (ROI) are boxed in white, and their magnified images are shown at the bottom. d The protein expression of IL-6 and TNF-α in liver tissue (up) and adipose (down) were evaluated by western blot. e The expression of IL-6 and TNF-α mRNA in liver tissue (left side) and adipose tissue (right side) were evaluated by RT-PCR. Data are presented as mean ± SEM (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, vs mice in MC group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs mice in EM group, p values were calculated by unpaired two-sided Student’s t-test. Scale bars, 200 μm (b, c bottom left) and 20 μm (b, c, up right)