Macrophage immunomodulatory activity of Acanthopanax senticousus polysaccharide nanoemulsion via activation of P65/JNK/ikkαsignaling pathway and regulation of Th1/Th2 Cytokines

Preparation of ASPS loaded k-Carrageenan beads(k-Car-ASPS)

k-Car-ASPS was prepared as follows, 0.1 g of k-Car was added into 50 ml of distilled water and the temperature was adjusted to 80 °C, the prepared contents were stirred using magnetic stirrer until k-Car was dissolved completely. A stock solution of curcumin was prepared in ethanol at a concentration of 1 mg/ml. In parallel 50 mg of ASPS was dissolved in 5 ml of aqueous solution and added to the above stirred solution. The solution was again re-stirred for 1 h at 80 °C to obtain a clear, viscous and homogeneous solution without bubble. Finally, the bead production process was carried out using the interphase technique. The above-prepared mixture containing Carrageenan and curcumin were dropped through a syringe into the beaker containing sunflower oil in aqueous phase (5% KCl). Beads were passed through the oil layer and it was collected in the aqueous phase;resulting in spherical beads left behind in the aqueous phase for an hour.

Preparation of ASPS-NE

The ASPS-NE formulations were synthesized using the method of pseudoternary phase diagram (Yu et al., 2020; Zhao et al., 2020). On the basis of the nanoemulsion regions in the phase diagram, ethanol was selected as the co-surfactant (Germany BASF Co., Ltd). Tween-80 and span-80 (Damao Chemical reagent factory, Shanghai, China) were selected as surfactants (Cao et al., 2019; Wang et al., 2018). Liquid paraffin (Damao Chemical reagent factory, Tianjin, China), Isopropyl myristate (IPM, Lanxi Wumart Chemical Co., Ltd., Zhejiang, China) was selected as the oil phase, Acanthopanax senticosus polysaccharide (ASPS: purity =98%, average MW =30.51 kDa) was provided by Shanghai Yuanye Biotechnology Co., Ltd. First, k-Car-ASPS was dissolved in deionized water, and the resulting aqueous solution was then mixed with ethanol and liquid paraffin, followed by the addition of respective amounts of Tween-80, Span 80, and IPM. The clear and transparent nanoemulsion was obtained after magnetic stirring. The final NE formulation comprised 46.2% Tween-80, 15.4% Span 80, 7.6% ethanol, 15.4% liquid paraffin, 11.5% IPM, and 3.9% aqueous ASPS solution.

Identification of the appearance and type

The appearance of the prepared ASPS-NE was identified in terms of homogeneity, transparency, and clarity. In addition, the ASPS-NE type was identified by adopting Methylene blue and Sudan red spreading speed methods (Solarbio, Beijing, China).

Identification of morphology and particle size

Transmission electron microscopy (Tecnai G220, USA) was employed to observe the shape of the prepared ASPS-NE. Prior to characterization, the ASPS-NE formulations were diluted in IPM to a final volume of 500 µL. The diluted nanoemulsion droplets were dried with filter paper, and then dropped 20 mg /ml phosphotungstic acid solution (pH = 7.4) and negatively stained on the copper net for 3 min. The nanoemulsion droplets were dried naturally. the morphology of nanoemulsion droplets was observed under TEM .This experiment was repeated in six times. The size distribution, polydispersity index (PDI), and zeta potential of the freshly prepared ASPS-NE formulations were determined using the NE particle size analyzer (Zetasizer 3000 H (Malvern Instruments Ltd, Worcestershire, UK).

In vitro release determination

The exact same amounts (calculated according to the content of ASPS) of ASPS-NE and ASPS were used. The dialysis tube was placed inside a hanging basket, which was placed in a dissolution cup containing 350 ml of PBS. The saline solution was maintained at (37 ± 0.5) °C and 100 r/min, and 3.5 ml samples were retrieved from the cup at regular intervals, along with the simultaneous supplementation of the released medium. The retrieved sample was diluted in PBS to a final volume of 7 ml, and the suspension was subjected to 20 min of centrifugation at 10,000 rpm and 4 °C. An ultraviolet spectrophotometer was employed to measure the absorbance (OD) value, followed by the calculation of the cumulative release percentage.

Stability

The cold–hot cycle experiment (4 °C, 60 °C), the lighted test (4500 ± 500 lx), and the accelerated stability test (40 ± 2 °C, 75% ± 5% RH) were conducted to evaluate the stability of the prepared ASPS–NE formulations.

Animals and immunizations

ICR mice (7-weeks-old, male, healthy,body weight (BW) 18∼22 g) were provided by the experimental Animal Center of Zhengzhou University. Each mice was subjected to one week of adaptive feeding prior to the initial immunization (license: SCXK (SU) 2020–0001),The mice were allowed to eat and drink freely, well ventilated, alternated between light and dark for 12h-12 h, and observed the health of the mice.

All animal experiments were conducted in accordance with the guidelines of the Animal Welfare and Ethics Committee (PZ2020003) and the care and use of laboratory animals (Henan University of Animal Husbandry and Economics IACUC; Approval ID: 2020BAD34B02). All mice were maintained climate controlled (temperature: 25 °C ± 1 °C; humidity: 40% ± 10%) and photoperiod controlled (12 h light–dark cycles) housing with free food and water.

Eighty mices were randomly divided into eight groups, and two mices per cage (n = 10). The specific grouping are as follows: blank group (NC), model group (MC, cyclophosphamide, 80 mg/kg/d), positive drug group (PC), ASPS-NE low-dose group (ASPS-NE(L), 31.25 mg/kg/d), middle dose group (ASPS-NE(M), 62.5 mg/kg/d), high-dose group (ASPS-NE(H), 125 mg/kg/d), NE group and ASPS group (62.5 mg/kg/d), respectively.

The specific administration methods are as follows: the pc group was injected with thymopentin (Beijing double-aigrettes; Pharmaceutical Co. Ltd.) at 10 mg/kg/d; ASPS-NE (H, M, L) groups were injected with ASPS-NE at 125, 62.5, 31.25 mg/kg/d , respectively; ASPS group was injected with ASPS at 62.5 mg/kg/d; the NE group was injected with NE at 0.2ml/d; the nc group was injected with normal saline at 0.2 ml/d. all the above groups were treated for 7 days. except for the nc group, all the other groups were injected with cyclophosphamide (CY, Beyotime Biotechnology Co., Ltd.) at a dose of 80 mg/kg/d on day 1,2.5, respectively.

The Mice was euthanized by pentobarbital (ip 100 mg/kg.w) after receiving the last dose and then put to death with cervical dislocation. Each animal was managed according to the guidelines of the local Ethics Committee and the Chinese law. Any anticipated or unexpected adverse events will be reported.

Splenocyte proliferation assay in vivo

Splenocyte activation was evaluated using the splenocyte proliferation assay as described in a previous study (Ren et al., 2017) .The animals were sacrificed 24 h after receiving the last dose to isolate splenocytes.

Freshly separated splenocytes (2.5 × 10⁶ cells/ml) from the seven groups were first cultured in RPMI-1640 solution (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), Concanavalin A (ConA, 10 µg /ml, Sigma Co.), or LPS (5 µg/ml) (Sigma Co.), and then inoculated into the wells of 96-well plates (100 µL/well) in triplicate. The plates were incubated at 37 °C in a 5% CO₂ atmosphere for 44 h. cell proliferation was estimated using the CCK-8 method. The average OD value was determined at 450 nm using a microplate reader (Varioskan; Thermo Scientific). Each experiment was repeated four times.

Spleen and thymus index measurements

The animals were sacrificed 24 h after receiving the last dose to isolate the thymus and spleen, and then the organs were weighed. The spleen and thymus indices were calculated using the following equation:

Spleen index (mg/10 g) = spleen weight (mg)/BW (mg) ×10

Thymus index (mg/10 g) = thymus weight (mg)/BW (mg) ×10

Macrophages activation

Macrophage activation was detected using the neutral red uptake experiment. The cells obtained from each group were washed with Hanks’ solution and then subjected to 0.1% neutral red staining for 2 h. Subsequently, 5  × 10⁴/ml macrophage cell suspension was added to each well of the 96-well plates, and the plates were incubated at 37 °C in 5% CO₂ conditions for 44 h. After the incubation, 100 µL of the neutral red solution was added to each well, followed by two washes with Hanks’ solution. Next, 100 µL of cell dissolution buffer (0.01% acetic acid and ethanol (1:1, v/v)) was added to each well for cell dissolution. Finally, the microplate (Varioskan; Thermo Fisher Scientific) was subjected to OD measurement at 540 nm. All the experiments were repeated four times.

ASPS-NE enhances the proliferation and differentiation of CD4⁺ and CD8⁺ T cells

The mice were sacrificed 24 h after receiving the last dose, and peripheral blood samples were collected in tubes containing the anticoagulant. In order to evaluate the proportions of CD3⁺CD4⁺/CD3⁺CD8⁺T cells, the cells were allowed to react with anti-CD3-APC, anti-CD4-PE, and anti-CD8a-FITC antibodies (BD, USA), followed by the analysis of the peripheral blood lymphocytes in a flow cytometer (Beckman CytoFLEX FCM).

Serum cytokine detection through ELISA

The serum levels of cytokines IFN-γ, TNF-α, IL-2, and IL-4 were determined using the corresponding ELISA kits (RD Co. Ltd.) in accordance with the manufacturer’s specifications. Each experiment was performed in triplicate.

Western Blot assay for P65/JNK/ikk α signaling pathway in peritoneal macrophages

The total protein in the peritoneal macrophages was extracted using the radioimmunoprecipitation assay (RIPA) lysis buffer (Boster Tech Co., Ltd, China) in accordance with the specific protocols recommended by the manufacturer. The protein contents were determined using the BCA method.

The proteins were separated by applying 40 mg of the protein lysate on an SDS-PAGE gel, and the separated proteins were transferred to 0.2-mm PVDF membranes. Subsequently, the membranes were blocked by incubation with Tris-buffered saline (TBS) supplemented with 5% bovine serum albumin (BSA) and 0.05% Tween 20 for 30 min under ambient temperature. Next, the blocked membranes were incubated overnight with primary antibodies dissolved in TBS supplemented with 1% BSA and 0.05% Tween-20. After the incubation and washing, the membranes were further incubated with horseradish peroxidase (HRP, 1/1000)-labeled secondary antibodies dissolved in TBS containing 1% BSA and 0.05% Tween-20 for 1 h under ambient temperature. After sufficient washing with TBS supplemented with 0.05% Tween 20, the ECL Western Blotting Detection Reagent (RPN2106, Amersham) was used to develop the protein blots for as long as 30 min. The ImageJ software (US National Institutes of Health) was employed to quantify the band intensity, with b-actin used as the loading reference.