Effect of Oroxylum indicum on hepatocellular carcinoma via the P53 and VEGF pathways based on microfluidic chips

Abstract

Background: Hepatocellular carcinoma (HCC), abbreviated as liver cancer, is one of the most common cancers in clinics. HCC has a wider spread and higher incidence due to its high malignancy and metastasis. In HCC, effective strategies to block cancer cell migration, invasion, and neovascularization need to be further studied. Consumption of flavonoid-rich Oroxylum indicum (OI) has been associated with multiple beneficial effects, including anti-inflammatory and anticancer properties, but the potential effects on HCC have not been thoroughly investigated.

Objective: In this study, we aimed to reveal the effect of OI on HCC and its potential mechanism through microfluidic technology.

Methods: We designed microfluidic chips for cell migration, invasion, and neovascularization to evaluate the effect of OI on HepG2 cells. To further explore the mechanism of its anti-liver cancer action, the relevant signaling pathways were studied by microfluidic chips, RT‒qPCR and immunofluorescence techniques. Compared to the control group, cell migration, invasion, and angiogenesis were significantly reduced in each administration group. According to the P53 and VEGF pathways predicted by network pharmacology, RT‒qPCR and immunofluorescence staining experiments were conducted.

Results: The results showed that OI upregulated the expression of Bax, P53 and Caspase-3 and downregulated the expression of Bcl-2 and MDM2. It has been speculated that OI may directly or indirectly induce apoptosis of HepG2 cells by regulating apoptosis-related genes. OI blocks the VEGF signaling pathway by downregulating the expression levels of VEGF, HIF-1α and EGFR and inhibits the migration and invasion of HepG2 cells and the formation of new blood vessels.

Conclusion: Our findings suggest that OI may inhibit the migration, invasion, and neovascularization of HepG2 cells, and its regulatory mechanism may be related to the regulation of the P53 and VEGF pathways.

Keywords: Anti-liver cancer; HUVEC; HepG2; Microfluidic chip; Oroxylum indicum.

Fig. 1

Cell migration chip The fluid channel layer has four identical modules, which can simultaneously screen a variety of anti-migration drugs of liver cancer cells, reflecting the integration and high efficiency of the microfluidic chip

Fig. 2

Cell invasion chip Cancer cells can penetrate human diaphragm growth, so this experiment adopts the descent of the chip of the polycarbonate membrane simulation of the artificial membrane to conduct research on OI HepG2 cell activity. Microfluidic chips can control the fluid in time and space at the same time and better simulate the real environment in vivo

Fig. 3

Cell neovascularization chips Channel 1 and channel 5 are the culture medium channels that can not only ensure the supply of nutrients but also reduce the impact of fluid flow on cells. Passage between the small rectangular structure can guarantee the precise localization of fluid infusion, can not only realize physical isolation of different kinds of cells, and can realize the mutual communication between cells, conducive to control the position of the gel distribution at the same time, in ensuring that the channel can be completely filled with liquid limit contents as needed in the channel at the same time, not easy to overflow. The matrix collagen solution was injected into channel 3 to fill the channel and act as a barrier to separate the two types of cells. HepG2 cells were encapsulated by Matrigel and injected into channel 2 to establish a 3D growth condition, and HUVECs were distributed in channel 4 as a 2D monolayer of adherent growth

Fig. 4

The chip for RT‒qPCR analysis The cell culture area can meet the number of sample cells used in the experiment. Cultured cells in the chip can make up for the traditional orifice or cultivation bottles, and other containers cannot be simulated in vivo. Building similar to the cells in the human body physiological micro space can further increase the reliability of the test results

Fig. 5

OI suppresses the migration of HepG2 cells a. OI reduces HepG2 cell migration b. Effect of OI on the relative migration of HepG2 cells ($\overline{x}\pm s$, n = 3). Compared with the Control group, ***P<0.001

Fig. 6

Effect of OI on the invasion of HepG2 cells a. OI reduces HepG2 cell invasion b. Effect of OI on the relative invasion rate of hepatocellular carcinoma HepG2 cells ($\overline{x}\pm s$, n = 3). Compared with the control group, ***P<0.001

Fig. 7

Detection of cell morphology a. Morphology of HepG2 cells in the culture model b. Morphology of HUVECs in the culture model

Fig. 8

Tube formation in HUVECs a. Control group b. Sorafenib group c. VEGF165 group; d. Low dose group of OI (0.2 mg·mL⁻¹) e. Middle dose group of OI (0.5 mg·mL⁻¹) f. High dose group of OI (0.8 mg·mL.⁻¹)

Fig. 9

Cavity area ratio compared with the control group, ^##P < 0.01. Compared with the VEGF₁₆₅ group^*P < 0.05, ^**P < 0.01 A. Control group B. Sorafenib group C. VEGF165 group D. Low dose group of OI (0.2 mg·mL⁻¹) E. Middle dose group of OI (0.5 mg·mL⁻¹) F. High dose group of OI (0.8 mg·mL.⁻¹)

Fig. 10

Relative expression of VEGF and HIF-1α compared with the control group, ^##P < 0.01. Compared with the VEGF₁₆₅ group^*P < 0.05, ^**P < 0.01 A. Control group B. Sorafenib group C. VEGF165 group D. Low dose group of OI (0.2 mg·mL⁻¹) E. Middle dose group of OI (0.5 mg·mL⁻¹) F. High dose group of OI (0.8 mg·mL.⁻¹)

Fig. 11

Construction of network a. PPI diagram: The bait proteins are marked by large blue nodes. Connecting lines show the number of interactions (direct or shared prey proteins) between bait genes. b GO function analysis c. KEGG function analysis

Fig. 12

Effect of OI on related gene expression in HepG2 cells A. Control group; B. Low-dose OI group; C. Medium-dose OI group; D. High-dose OI group. Compared with the control group, ^*P<0.05, ^**P<0.01

Fig. 13

Immunofluorescence staining The immunofluorescence staining method of secondary antibody indirect staining labeled by FITC was adopted, which can stimulate green fluorescence at 488 nm wavelength with high sensitivity and flexible staining. OI may inhibit the progression of liver tumors by regulating P53 and VEGF expression

Fig. 14

Mechanism of OI in the treatment of liver cancer. OI inhibits the migration, invasion and neovascularization of HepG2 cells by activating the P53 pathway and blocking the VEGF pathway