Part 2 The Phenylethanol Glycoside Liposome Inhibits PDGF-Induced HSC Activation Via Regulation Of The FAKPI3KAkt Signaling Pathway
Mar 06, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
4. Materials and Methods
4.1. Materials
CPhGs (phenylethanoid glycoside from cistanche) were purchased from Hetian Di Chen Medical Biotechnology Co., Ltd. (Xinjiang, China). The content of the echinacea was more than 35%, and that of the acteoside was more than 16%. Lecithin was purchased from Shanghai Lanji Technology Co., Ltd. (Shanghai, China). 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and cholesterol were supplied by Avanti Polar Lipid Inc. (Birmingham, AL, USA). An immortalized rat hepatic stellate cell was provided by Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) was bought from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (Waltham, MA, USA). Penicillin (100 卩g/mL) and streptomycin (100 卩g/mL) were products of Thermo Fisher Scientific, Inc. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (5 mg/mL) was purchased from Sigma (Oakville, ON, Canada). LDH activity was assessed by using an LDH assay kit from Beijing Solar Science & Technology Co., Ltd. (Beijing, China). The FITC Annexin V Apoptosis Detection Kit I and propidium iodide (PI)/RNase Staining Buffer were all purchased from BD Biosciences (San Diego, CA, USA) and rrPDGF-BB was provided by R&D Systems (Minneapolis, MN, USA). The TRIzol reagent was bought from Thermo Fisher Scientific, Inc. The RevertAid First Strand cDNA Synthesis kit was the product of Thermo Fisher Scientific, Inc. (Vilnius, Lithuania). TB Green Premix Ex Taq TM II was purchased from Takara (Dalian, China). Ripa pyrolysis solution was provided by Thermo Fisher Scientific, Inc. Protease inhibitor phenylmethylsulfonyl fluoride and the Broad-spectrum phosphatase inhibitor mixture were bought from Boster Biological Technology Co., Ltd. (Wuhan, China). Protein bands were visualized by a Pierce™ ECL Western Blotting Substrate, which was provided by Invitrogen (Carlsbad, CA, USA). The horseradish enzyme labeling goat anti-rabbit IgG was purchased from Zhongshan JinQiao Shanghai Co., Ltd. (Shanghai, China). The Super Signal West Femto Trial Kit was supplied by Thermo Fisher Scientific, Inc. Rabbit anti-p-actin, a-SMA, collagen-1, FAK, PI3K, phospho-PI3K, Akt, and the phospho-Akt antibody were all purchased from Beijing Bioss Antibodies Biological Co., Ltd. (Beijing, China). The X-3-FAK and X-3-NC were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The Lipofectamine 2000 was provided by Invitrogen.
4.2. Preparation of the CPhG Liposomes
Lecithin, DPPC, and cholesterol (1:2:2, m/m/m) were dissolved in a mixture of chloroform and methanol (2:1 v/v), rotated to form a film at 49-50 °C, and CPhGs (phenylethanoid glycoside from cistanche)(250 卩g/mL) were added subsequently. The reactive solution was rotated and hydrated continuously, and the phospholipid membrane was transferred into the water to form CPG liposomes. The encapsulated liposome process was repeated according to the above methods. To obtain a uniform liposome, the encapsulated liposome was extruded 5 times with a 0.45 卩m microporous membrane and filtrated 18-20 times with a 0.22 卩m membrane. The CPG liposomes were stored at 4 °C. The particle size distribution was measured by a laser particle size analyzer, the particle shape was observed by transmission electron microscopy, and the drug loading and encapsulation efficiency were determined by HPLC.
4.3. In Vitro Release from the CPG Liposome and CPhGs(phenylethanoid glycoside from cistanche)
The in vitro release profile of the CPG liposomes and CPhGs(phenylethanoid glycoside from cistanche) was carried out using the dialysis membrane method. A total of 5 mL of each sample was transferred into a dialysis bag with a 14,000 Da molecular weight cut-o任 The dialysis bag was immersed into 200 mL of PBS buffer solution (pH = 6.0) vibrated at 37 °C. The release medium (3 mL) was withdrawn and replaced with an isopycnic PBS buffer solution (pH = 6.0) at scheduled time intervals (0.5,1, 2, 4, 8,12,16, 24, and 48 h). The concentrations of the echinacoside in the CPG liposomes and CPhGs in the release medium were determined by
HPLC. The cumulative release (%) of the CPG liposomes and CPhGs(phenylethanoid glycoside from cistanche) was calculated by using the following Equation (1):
where Cn is the drug concentration in the release medium at the nth sampling point, V is the volume of the released medium, and W is the total dose.
To better understand the vitro release characteristic of the CPhG(phenylethanoid glycoside from cistanche) liposome and CPhGs, three different models were applied to fit the obtained release data: (a) the zero-order kinetics model, (b) the first-order kinetics model, and (c) the Higuchi model given in Equations (2)-(4):
where Mt/Mo is the fraction of curcumin released from the sample at time t, and k is the release constant of the different models. The correlation coefficient is calculated to illustrate the fitting degree between the release model and the data.
4.4. Cytotoxicity
For the HSCs, a density of 1 x 105 cells/mL was plated in 96-well plates and allowed to grow overnight at 37 °C in a 5% CO2 incubator. Cell vitality was tested on a wide range of CPhG liposome concentrations (117.79,58.90, 29.45, 14.72, 7.36, 3.68,1.84, 0.92, and 0.46 卩g/mL) to detect the test cells vitality and calculate the IC50 values. Then, the cells were incubated at 37 °C for 4 h with 20 卩L of MTT (5 mg/mL in PBS solution). The purple MTT-Product product was dissolved in a 150 卩L DMSO, and a Multiskan Spectrum Absorbance Reader (Fisher Scientific, Inc., Waltham, MA, USA) was used to measure the absorber at 490 nm. The following formula (Equation (5)) was used to calculate the percentage of cell viability:
4.5. Cell Toxicity ofthe CPhG Liposomes on HSCs
Cells were seeded into 6-well plates, and the toxicity of the HSCs was measured using an LDH detection Kit. Cells were plated at a density of 2 x 105 cells/well in 6-well plates at 37 °C in an incubator with 5% CO2. After overnight growth, cells were treated with CPhG liposomes (29.45, 14.72, and 7.36 卩g/mL). A blank (DEME media) control was maintained simultaneously. After 24 h, the cells were collected by trypsin and centrifugation. Each group of cells was administered strictly in accordance with the kit instructions. The LDH leakage rate of the cell culture fluid was then calculated.
4.6. Apoptosis Analysis
Cells were cultured into 6-well plates with a density of2 x 105 cells/well at 37 °C in an incubator with 5% CO2. After overnight growth, cells were treated with CPhG liposomes (29.45, 14.72, and 7.36 卩g/mL), and then the apoptosis of HSCs was measured using the FITC Annexin V Apoptosis Detection Kit. The concentrations were based on the IC50 determined from the cytotoxicity assay. A blank (DEME media) control was maintained simultaneously. After 24 h, the cells were collected by trypsin and centrifugation and washed with PBS and suspended in a 500 卩L binding buffer. Then, the cells were stained with 5 卩L Annexin V-FITC and 5 卩L PE. Cells were incubated at room temperature for 30 min in the dark, and the apoptotic rate was measured using a FACSCalibur Cytometer (Becton Dickinson, San Jose, CA, US).
4.7. Cell Cycle Arrest Analysis
HSCs (2 X 105 cells/2 mL/well) were treated for 24 h with CPhG liposomes at 29.45, 14.72, and 7.36 ^g/mL in 6-well plates. A blank (DEME media) control was maintained simultaneously. The cells were harvested and washed with 1 mL of PBS and fixed with 75% ethanol overnight at 4 °C. The cells were then centrifuged and resuspended in 500 solutions of propidium iodide (PI)/RNase staining buffer at 37 °C for 30 min in the dark. Finally, the cells were measured using a FACSCalibur Cytometer (Becton Dickinson). The proportions of the cells in the G0/G1, S, and G2/M phases of the cell cycle were analyzed by the FACS Diva software (Becton Dickinson).
4.8. Effect of the CPhG(phenylethanoid glycoside from cistanche) Liposome on the PDGF Mediated FAK and PI3K/Akt Signaling Pathway
4.8.1. Proliferation Assay
The HSC lines (1 X 105 cells/mL) were plated in 96-well plates and allowed to grow overnight at 37 °C in a 5% CO2 incubator. After 24 h of cell culture, the cells were randomly divided into the following five groups: A blank control (cultured in DMEM containing 10% FBS only), PDGF-BB (cultured in DMEM containing 50 卩g/L PDGF-BB), a CPhG liposome intervention (stimulated with medium containing 50 昭/mL PDGF-BB 24 h). Then, the cells were treated with 29.45,14.72, and 7.36 Rg/mL of CPhG liposomes. The cells were continuously cultured for 24 h, 48 h, and 72 h in the incubator at 37 °C for 4 h with 20 rL ofMTT (5 mg/mL in PBS solution). The purple MTT-formazan product was dissolved in 150 rL of DMSO and estimated by measuring the absorbance at 490 nm in a Multiskan Spectrum Absorbance Reader (Thermo Fisher Scientific, Inc.), and the IC50 was calculated. Quadruplicate samples were run for each concentration in four independent experiments. The following formula (Equation (6)) calculated the percentage of cell viability:
4.8.2. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Cells were seeded into 6-well plates with a density of 1 X 105 cells/mL. This grouping method is the same as the previous one. After 24 h, the total RNA was extracted from the cultured cells by a TRIzol reagent. The concentration and purity of the RNA were measured at 260/280 nm using an ultraviolet spectrophotometer according to the standard OD260/OD280 ratio of 1.8-2.0. The reverse transcription reactions were reversely transcribed from the RNA using the RevertAid First Strand cDNA Synthesis kit. A TB Green Premix Ex TaqTM II was used to measure gene expression. The qRT-PCR was performed using an ABI QuantStudio™6 Flex Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Primer sequences were synthesized by Sangon Biotech (Shanghai, China) (Table 4).
4.8.3. Western Blot Analysis
The total proteins extracted from HSCs in different groups were prepared as described previously. Briefly, the similar proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics GmbH, Mannheim, Baden-Wuerttemberg, Germany). Then, the PVDF membranes were blocked in 3% BSA and incubated overnight at 4 ◦C with the primary antibodies. The next day, HRP-labeled goat anti-rabbit IgG (1:25,000 dilution; 1 h) was added. Protein bands were visualized via a Pierce™ ECL Western Blotting Substrate and imaged using the FluorChem E Imaging System (Protein Simple, San Francisco, CA, USA), which were normalized with β-actin as an internal control.
4.8.4. FAK Overexpression Experiments
The cells were divided into the normal medium control group, the PDGF-BB stimulation group, the PDGF-BB stimulation + X-3-FAK group, the PDGF-BB stimulation + X-3-NC group, the PDGF-BB stimulation + X-3-FAK + CPhG liposome group, and the rrPDGF-BB stimulation + X-3-NC + CPhG liposome group. The HSC lines (1 × 105 cells/mL) were plated in 6-well plates and allowed to grow overnight at 37 ◦C in a 5% CO2 incubator. On the next day, PDGF-BB (cultured in DMEM containing 50 µg/L PDGF-BB) cells were added and cultured. On the third day, pEX-3-FAK and X-3-NC were diluted in an antibiotic-free medium, mixed well with lipofectamine 2000, and allowed to stand at 37 ◦C for 20 min, before being added to each well of the cells at 37 ◦C. After being incubated for 5 h, the medium containing 10% serum was replaced for 48 h, and on the fifth day, the medium was replaced with the concentration of 29.45 µg/mL CPhG liposome for 24 h. Then, each well was collected on the second, third, fifth, and sixth days, and the expressions of the FAK, PI3K, Akt, p-PI3K, and p-Akt gene proteins were detected.
4.9. Statistical Analysis
Data were expressed as the means ± standard deviation (SD). The SPSS 16.0 software (IBM, New York, NY, USA) was used for statistical analyses. The significance of the difference was calculated by a one-way ANOVA test and followed by an LSD test. The drug dissolution equation was fitted by Origin 8.5 (OriginLab, Northampton, MA, USA).
5. Conclusions
In conclusion, our results demonstrate that CPhG(phenylethanoid glycoside from cistanche) liposomes significantly inhibit HSCs activation, markedly attenuating the development of liver fibrosis by increasing apoptosis and regulating the cell cycle in HSCs. Inhibiting the activation of FAK/PI3K/Akt signaling pathways may be the underlying mechanism by which the CPhG liposomes protect against chronic liver disease associated with fibrosis. These findings provide novel insights into the mechanisms of this new drug formulation as an antifibrogenic candidate for the future treatment of hepatic fibrosis. However, the underlying mechanisms are more complex than what is described here, and our results do not exclude the possible involvement of other devices caused by the CPhG liposomes to treat liver fibrosis.
Author Contributions: T.L., J.Z., S.-P.Y., L.M., and S.-L.Z. conceived and designed the experiments. S.-L.Z., S.-P.Y., T.L., J.Z., X.-T.M., and X.-Y.Y. analyzed the data. S.-L.Z. and J.Z. wrote the manuscript. T.L., L.M., and J.Z. reviewed the manuscript. All authors read and approved the final manuscript.
Funding: This research was funded by the National Natural Science Foundation of China, grant number 81560628.
Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (No. 8156140127). The authors would like to express their sincere thanks to Jun Zhao and Xin Wang for their improvement of the writing of this paper.
Conflicts of Interest: The authors report no declarations of interest.
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