The therapeutic effect of ultrasound targeted destruction of schisandrin A contrast microbubbles on liver cancer and its mechanism

Ultrasonic cell crushing instrument (Ningbo Xinzhi Biological Technology Co., LTD.), Doppler ultrasound diagnostic instrument (Kunshan Ultrasonic Instrument Co., LTD.), Zeta potential/particle size instrument (British Malvern Instrument Co., LTD.), SW-CJ-1D single side vertical air supply purification table (Suzhou Zhijing Purification Equipment Co., LTD., Jiangsu Province), HZQA Constant temperature incubator (Jintan Shenglan Instrument Manufacturing Co., LTD.), LX-C50L vertical automatic electric heating pressure steam sterilizer (Beijing Sibo Shengda Technology Co., LTD.), scanning electron microscope (Japan Electronics Co., LTD.), Ultrasound imaging instrument (mindray M9cv, Superficial probe), Bio-Rad 680 iMark Microplate reader (American Bio-Rad Co., LTD.)

Span60 (Tianjin BASF Chemical Co., LTD.), PEG1500 (Tianjin Guangfu Fine Chemical Research Institute), NaCl (Tianjin Beilian Fine Chemical Development Co., LTD.), schisandrin A (Chengdu Manst Biotechnology Co., LTD. HPLC ≧ 98%), phenol and sulfuric acid (Tianjin Kemio Chemical Reagent Co., LTD.), Walker-256 (number: 399-88-2) was obtained from Shanghai Hongshun Biotechnology Co., LTD. MEM (containing NEAA) basal medium (Procell PM150410) and fetal bovine serum (Procell 164210) was obtained from Pricella Biotechnology Co., LTD. Fixative solution (4% Paraformaldehyde, P1110) was obtained from Shanghai solarbio Bioscience & TechnologyCo., LTD. Enzyme linked immunosorbent assay (ELISA) kit tumor necrosis factor-α (TNF-α) (ab236712), interleukin-1β (IL-1β) (ab255730) and interleukin-6 (IL-6) (ab234570) was obtained from abcam Bioscience & TechnologyCo., LTD. Hematoxylin-eosin (HE) Stain Kit (G1120) was obtained from Solarbio Bioscience & TechnologyCo., LTD. Immunohistochemistry kit hypoxia inducible factor-1α (HIF-1α) (IHC0103715), VEGF(IHC0100011) and vascular endothelial growth factor receptor 2 (VEGFR-2) (IHC0102817) was obtained from Shanghai CaiYOU industrial Co., LTD. WB kit: Primary antibody p-PI3K (bs-6417R), PI3K(20584-1-AP), p-Akt (bs-0876R) was obtained from Bioss Co., LTD, AKT (60203-2-Ig), p-mammalian target of rapamycin (mTOR) (67778-1-Ig), mTOR (66888-1-Ig), was obtained from Proteintech Group, GAPDH was obtained from Hangzhou Hua ‘an Biotechnology Co. LTD, Secondary antibody (SA00001-1) was obtained from Bioss Co., LTD.

Walker-256 cell (Free of mycoplasma infection, cells were derived from ascites of liver cancer in rats) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37°C in 5%CO₂ incubator. The cells were routinely digested and subcultured with 2.5 g/L trypsin, and the logarithmic growth phase cells were used for experiments.

36 SD (Sprague Dawley, male, 6 weeks, 180−200 g, wide type rats) rats were obtained from Henan Laboratory Animal Center. Animal experiment ethics was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. KY2023-006).

450 mg Span60, 900 mg NaCl, 450 mg PEG-1500 and 300 mg schisandrin A were weighed, placed in a mortar and thoroughly ground, dissolved in 40 mL PBS phosphate buffer solution, and heated to 80°C in a magnetic heating mixer, and stirred and dispersed evenly. Then, the solution was continuously sonicated at 570 W power for 6 min using an ultrasonic cell disruptor by acoustic cavitation method, while nitrogen gas was continuously injected into the above solution. A uniform milky yellow liquid mixture was prepared and centrifuged in an ultracentrifier at 2 000 g for 8 min. After centrifugation, a stratified solution was obtained. The upper and middle layers were removed and placed in a 250 mL separating funnel, washed with an equal volume of PBS phosphate buffer, and left to stand. The middle layer microbubbles were collected and freeze-dried to obtain Span-PEG ultrasound contrast agent microbubbles loaded with schisandrin A.¹⁴ The size and shape of the microbubbles were observed by scanning electron microscopy (SEM). Detailed operation details were as follows: A small amount of microbubble powder was coated to one side of the double-sided glue and the other side was fixed on the stage of the scanning electron microscope. The surface morphology of the drug microbubbles was observed and photographed by scanning electron microscopy under a high voltage of 15 kV at a magnification of 5000. The particle size distribution and Zeta potential of the microbubbles were determined by ZS90 laser particle size analyzer. Detailed operation steps were as follows: Added pure water into the sample tank as the dispersing agent, turned on the ultrasonic disperser and set the intensity to 7, turned on pump switch after 2 minutes, adjusted the pump speed to 2680 r/min, specified water as the dispersing agent in the TAB, and other parameters were determined, clicked “Start”, measured the sample, and saved the results. Then changed the measurement conditions and re-measured until the next measurement results were basically in line with the last measurement results, then the last measurement results are the particle size measurement results of the sample. This experiment was independently repeated three times with consistent results.

The loading rate of schisandrin A in Span-PEG microbubbles was determined by ultraviolet spectrophotometric method. The standard of schisandrin A was prepared at concentrations of 2.5 μg·mL⁻¹, 5 μg·mL⁻¹, 10 μg·mL⁻¹, 20 μg·mL⁻¹, 30 μg·mL⁻¹, 40 μg·mL⁻¹ and 50 μg·mL⁻¹, respectively. The absorbance value was measured at 254 nm, the standard curve was drawn, and the regression equation was calculated. Span-PEG microbubbles loaded with schisandrin A were weighed 10 mg, dispersed in distilled water for ultrasonic release for 2 h, filtered, and constant volume to a 10 mL volumetric flask. The absorption wavelength of schisandrin A in Span-PEG composite microbubbles at 254 nm was determined by the same method. The schisandrin A loading rate was calculated according to the standard curve. This experiment was independently repeated three times with consistent results.

The standard Schisandandin A was diluted in double dilutions in cell culture medium, so that the final concentrations in cell culture medium were 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL and 0 μg/mL, respectively. Walker-256 cells with logarithmic growth were seeded in 96-well plates and divided into eight groups. And culture medium containing the corresponding concentration of drug was added to each well (1×10⁴/mL), the cells and the drug were incubated in an incubator for 72 h. Then, 10 μL of freshly prepared 5 mg/mL MTT solution was added to each well and continue to culture for 4 hours. The supernatant was discarded and dissolved by adding 200 μL dimethylsulfoxide (DMSO), and the absorbance value was measured at 490 nm by microplate reader. The drug concentration in the control group was zero. In the blank group, only culture medium, MTT and DMSO were added. The cell survival rate = (Experimental group OD - blank group OD)/(Control group OD - blank group OD). The 50% inhibitory concentration (IC50) was calculated by IC50 software to define the concentration range of schisandrin A in this experiment.¹⁶ Fitting and calculation of cell survival curves were calculated with GraphPad Prism 9.0.

The above MTT assay was also used to detect following cell survival rate in control (C), microbubbles (M), ultrasound (U), drug (D), drug + ultrasound group (D+U), drug + microbubbles + ultrasound group (D+M+U) group. This experiment was independently repeated three times with consistent results.

The cells in logarithmic growth phase were used for the experiment and randomly divided into 6 groups: simple microbubbles group (M): the cells were resuspended in cell culture medium containing 5% microbubbles; simple ultrasound group (U): the cells were exposed to 300 kHz, 0.25 W ultrasound for 10 s; simple drug group (D): the cells were resuspended in cell culture medium containing 2.5 μg/mL schisandrin A; Drug + ultrasound group (D+U): The cells were incubated with 2.5 μg/mL schisandin A and then exposed to 300 kHz, 0.25 W ultrasound for 10 s; Drug + microbubbles + ultrasound group (D+M+U): After routine digestion and centrifugation, the cells were added with cell culture medium containing 2.5 μg/mL Schisandandin A and 5% microbubbles, and then ultrasound irradiation was performed under the same conditions as the previous group. Control group (C): The digested and centrifuged cells were resuspended and cultured in conventional culture medium.¹⁷ Cell survival rate was measured by MTT assay. This experiment was independently repeated three times with consistent results.

Sample collection: The treated cells of each group were cultured in 24-well plates with 6 multiple wells in each group. After 24 hours of culture, the cells of each group were routinely digested, centrifuged and collected, rinsed 3 times with PBS, and the last time was fixed in 1 mL double distilled water. Cells were destroyed using an ultrasonic cell morcellator to obtain a double distilled aqueous solution of intracellular fluid in each group. The chromatographic conditions were as follows: Kro-masil C18(250 mm×4.6 mm, 5 μm) column; The mobile phase was methanol-0.1% glacial acetic acid aqueous solution (82:18). Flow rate :1mL/min; Detection wavelength: 254 nm; The column temperature was 30°C. The chromatogram of schisandrin A samples and intracellular liquid standard was drawn under the selected chromatographic conditions.¹⁸ This experiment was independently repeated three times with consistent results.

After 7 days of adaptive feeding, all groups of animals were inoculated with Walker-256 cells in the liver under ultrasound guidance to establish an orthotopic liver cancer model. When the tumor gradually grew to 35mm², the drug treatment was started. The rats were divided into 6 groups with 6 rats in each group: control group (injected with equal volume of normal saline), microbubble group (injected with microbubbles via the tail vein without ultrasound), ultrasound group (injected with equal volume of normal saline with ultrasound), ultrasound + schisandrin A group (injected with schisandrin A via the tail vein), ultrasound + microbubble group (injected with microbubbles via the tail vein), and ultrasound + schisandrin A microbubble group (injected with schisandrin A microbubble via the tail vein). The liver parts of all animals were subjected to low-frequency ultrasound treatment at 300 kHz, 2.0 W/cm², PRF(Pulse Repetition Frequency) 1 kHz, DC(Duty Cycle) 20%, PNP(Peak Negative Pressure) 0.5 MPa, with 10 s irradiation, 10 s interval, and a total of 20 min. The injection dose of schisandrin A was 20 mg/kg, and the microbubble injection dose was 0.3 mL/kg. The treatment was given every 3 days for a total of 16 days. Tumor observation: Ultrasound was used to observe the tumor growth at the inoculation site 7 days and 16 days after treatment. This experiment was independently repeated three times with consistent results.

In this study, mindray M9cv Doppler ultrasound was used to collect the in situ tumor images of animals in each group before and after treatment. The acquisition method was as follows: under the guidance of ultrasound, the tumor site was determined, and the longest and shortest diameters of the tumor were measured at the same time. This experiment was independently repeated three times with consistent results.

Twenty-four hours after the last treatment, blood samples were collected from the abdominal aorta of rats and stored in a test tube without anticoagulant. The samples were placed at 37°C for coagulation, and after blood coagulation, the samples were equilibrated and centrifuged (4°C, 2000 g, centrifugal radius was 7.5 cm), and the final obtained supernatant was the serum. The contents of TNF-α, IL-1β and IL-6 in serum were determined by enzyme-linked immunosorbent assay. The specific operation steps are as follows: 10 μL standards and 10 μL samples were added into the wells of the corresponding reaction plates. 40 μL TNF-α/IL-1β/IL-6 Biotin and 40 μL TNF-α/IL-1β/IL-6 POD were added to each well. The plates were mixed gently for 30 seconds, the wells were sealed, and the plates were incubated at room temperature for 45 minutes. Washed the plate: dump all the liquid in the plate, washed the reaction plate with washing solution (add 350 μL of washing solution to each well), and removed water droplets (pat dry on thick absorbent paper): washed 5 times repeatedly. 100 μL of chromogenic solution was added to each well, gently mixed for 10 seconds, and incubated at room temperature for 20 minutes. Added 100 μL stop solution to each well. Gentle mixing for 30 s: OD values were read at 50 nm within 30 min. This experiment was independently repeated three times with consistent results.

Fresh liver tumor tissues were taken and fixed with fixative solution for more than 24 hours. The tissues were removed from fixative solution and trimmed in a fhood with a scalpel. Then, the tissues were dehydrated and immersed in wax, embedded, sectioned, hematoxylin staining, eosin staining, dehydrated and sealed. Finally, microscopic examination and image acquisition and analysis were performed. Pathological evaluation was made by pathologists under a microscope. Sample preparation was performed as follows: sampling, fixation, dehydration, transparency, wax immersion, embedding, and sectioning. The procedure for HE staining was as follows: The sections were successively washed in xylene I 10 min, xylene II 10 min, absolute ethanol I 5 min, absolute ethanol II 5 min, 95% alcohol 5min, 90% alcohol 5 min, 80% alcohol 5 min, 70% alcohol 5 min, distilled water. The sections were stained with Harris hematoxylin for 3−8 min, washed with tap water, differentiated in 1% hydrochloric acid alcohol for 7 seconds, rinsed with tap water, returned to blue in 0.6% ammonia water, and rinsed with running water. Sections were stained in eosin staining solution for 1-3min. The slices were successively placed in 95% alcohol I 5, min, 95% alcohol II 5 min, absolute ethanol I 5 min, absolute ethanol II 5 min, xylene I 5 min, xylene II 5 min for dehydration and transparency. The slices were taken out of xylene to dry slightly and sealed with neutral gum. This experiment was independently repeated three times with consistent results.

The paraffin sections of liver tumor tissue were deparaffinized to water, and then underwent antigen repair, endogenous peroxidase blocking, serum blocking, primary antibody, secondary antibody, DAB staining, nucleus counterstain, dehydration and sealing. Finally, the sections were taken out of xylene to dry slightly, and sealed with sealing glue for microscopic examination. Specific IHC procedures are as follows: Paraffin sections were routinely deparaffinized to water. 3% hydrogen peroxide was used for 10 min at room temperature to inactivate endogenous enzymes, and the cells were washed twice with distilled water. The sections were immersed in 0.01 mol/L citrate buffer solution (pH 6.0), heated to boiling in microwave oven, and then turned off. After an interval of 5 min, the sections were cooled repeatedly and washed twice × 3 min with PBS buffer (pH 7.2−7.6). The blocking solution was added droppers at room temperature for 20min. Toss off any excess liquid without washing. Appropriate primary antibodies (Rat Anti-VEGF 1:300; Rat Anti-VEGFR2 1:200; Rat Anti-HIF-1a 1:200), incubated at 37°C for 2 h. Washed 3 min x 3 times with PBS buffer (pH 7.2−7.6). The working solution of biotinylated secondary antibody was added at 20~37°C for 20min. Washed 3 min × 3 times with PBS buffer (pH 7.2−7.6). Working solution of horse-radish enzyme-labeled streptavidin was dropped and washed 3 min × 3 times with 20~37°C, 20 min, PBS buffer (pH 7.2−76). The reaction time was controlled under the DAB chromoscope at room temperature, generally between 5 and 30 min. Light counterstained with hematoxyli n, dehydrated, sealed with transparent neutral gum, and observed under microscopy. This experiment was independently repeated three times with consistent results.

The tumor tissue was taken and detected by Western blottting. About 200 mg of fresh tumor tissue was weighed, then liquid nitrogen was added while grinding in a mortar, the ground tissue was weighed in a peeled and precooled centrifuge tube. Then, the tumor tissue was fully lysed with RIPA (Radio Immuno Precipitation Assay) lysate containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and inhibitors containing sodium orthovanadate, sodium fluoride, EDTA, leupeptin. The total protein was extracted and the protein content was determined by BCA method. The protein samples (50 μg) were denatured, subjected to 12% polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) films, and blocked with 5% skim milk for 2 h at 4°C. Then, antibodies against PI3K, p-AKT, AKT, p-AKT, mTOR and pmTOR proteins were added (diluted 1:1 000) and incubated at 4°C overnight. The membranes were washed with TBST buffer, the secondary antibody (dilution 1:10 000) was added, and the membranes were incubated for 2 hours at room temperature. The membranes were washed with TBST (Tris Buffered Saline Tween20) buffer, and after adding ECL (Electro Chemi Luminescence) solution, the membranes were exposed and developed in an automatic chemiluminescence gel imaging analysis system. This experiment was independently repeated three times with consistent results.

SPSS 22.0 statistical software was used for all statistical analyses of this study. The measurement data were expressed as mean ± standard deviation. Comparison between groups was performed using analysis of variance. P < 0.05 for the difference was considered statistically significant.

Figure 1A shows the structure SEM of Span-PEG composite microbubbles loaded with schisandrin A. The prepared microbubbles have smooth surface and uniform particle size. Figure 1B and 1C show the particle size distribution and Zeta potential of Span-PEG composite microbubbles, respectively. From the measurement results and distribution curves, it can be seen that the composite microbubbles have uniform size and single peak particle size distribution. The average particle size is 595.3 nm, less than 700 nm, and the Zeta potential is −18.8 mV, which is relatively stable. It meets the requirements as an ultrasound contrast agent.

FIGURE 1.

According to the experimental method, the standard curve of schisandrin A was drawn. The standard curve equation of schisandrin A was: Y = 0.0121X-0.0012, R² = 0.9992.

The absorbance value of schisandrin A in Span-PEG composite microbubbles was measured and substituted into the standard curve to calculate the loading rate of schisandrin A. The results are shown in Table 1. The loading rate of schisandrin A in Span-PEG composite microbubbles was 8.84 ± 0.14%, the encapsulation efficiency was 82.24 ± 1.21%.

The results of MTT assay showed that the cell survival was inhibited to varying degrees after treatment with different concentrations of schisandrin A for 24h. The cell survival rates of 0 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, 16 μg/mL, 32 μg/mL, 64 μg/mL schisandrin A on Walker-256 cells after 24 hours were 100.00%, 72.36%, 64.52%, 45.90%, 33.39%, 24.78%, 21.97%, 17.44%, respectively. The IC50 value was 2.87 μg/mL. The concentration of schisandrin A selected for this experiment was 2.5μg/mL, as shown in Figure 2.

FIGURE 2.

Compared with the control group, the cell survival rate of each experimental group decreased significantly: group C (100%) > group U (98.31%) > group M (95.68%) > group D (53.14%) > group U+D (42.53%) > group U+M+D (32.17%). There were significant differences among the groups (F = 626.5, P < 0.0001), group D vs. group D+U (MD = −0.32587, P < 0.001), group D vs. group D+M+U (MD = −0.52608, P < 0.001), group D+U vs. group D+M+U (MD = −0.32673, P < 0.001), as shown in Figure 3.

FIGURE 3.

Under the selected chromatographic conditions, the chromatographic profiles of schisandrin A standard, blank intracellular liquid, blank intracellular liquid + schisandrin A standard and the tested intracellular liquid were obtained (Figure 4A−D). The standard curve of schisandrin A was drawn to obtain the linear regression equation Y = 0.0544X+0.0128 (R² = 0.9997), and the linear range was 0−6.4 μg/mL. The average concentration of schisandrin A in each group was calculated by taking the peak area of schisandrin A into the equation: group M (0 μg/mL); group U (0 μg/mL); Group D 0.33 μg/mL; Group D+U 0.46 μg/mL; Group D+M+U 0.76 μg/mL. There were significant differences in intracellular drug concentrations among the groups (F = 587.5, P < 0.0001), Group D vs. Group D+U (MD = −0.13667, P < 0.001), Group D vs. Group D+M+U (MD = −0.43500, P < 0.001), Group D+U vs. Group D+M+U (MD = −0.29833, P < 0.001), shown in Figure 4E.

FIGURE 4.

The experimental results showed that the tumor in the control group (without treatment drugs), M (microbubbles) group and U (ultrasound) group had progressed and enlarged, and the other different treatment groups had significant effects before and after drug treatment (P < 0.05). Among them, the tumors in the ultrasound + schisandrin A group and the ultrasound + microbubble group showed a certain degree of atrophy (P < 0.05). The most significant effect was in the ultrasound + schisandrin + A microbubble group (P < 0.01). The results suggest that schisandrin A has a certain anti-tumor effect, and the microbubbles loaded schisandrin A can promote the intake of schisandrin A in tumor cells after blasting at the tumor site under ultrasound irradiation, thus playing the best anti-tumor effect, as shown in Figure 5.

FIGURE 5.

The experimental results showed that the levels of inflammatory factors in the control group (without therapeutic drugs) were higher, and there was no significant improvement for the levels of inflammatory factors in M group and U group. However, there were significant differences among the groups for TNF-α, (F = 73.698, P < 0.001), Group C vs. Group D+U (MD = 0.745, P < 0.01), Group C vs. Group M+U (MD = 1.228, P < 0.01), Group C vs. Group D+M+U (MD = 2.060, P < 0.01), Group D+U vs. Group M+U (MD = 0.483, P < 0.01), Group D+U vs. Group D+M+U (MD = 1.315, P < 0.01), Group M+U vs. Group D+M+U (MD = 0.831, P < 0.01). There were significant differences among the groups for IL-6, (F = 828.16, P < 0.001), Group C vs. Group D+U (MD = 47.280, P < 0.01), Group C vs. Group M+U (MD = 68.040, P < 0.01), Group C vs. Group D+M+U (MD = 74.818, P < 0.01), Group D+U vs. Group M+U (MD = 20.760, P < 0.01), Group D+U vs. Group D+M+U (MD = 27.539, P < 0.01), Group M+U vs. Group D+M+U (MD = 6.78, P < 0.01). There were significant differences among the groups for IL-1β, (F = 230.955, P < 0.001), Group C vs. Group D+U (MD = 9.99, P < 0.01), Group C vs. Group M+U (MD = 17.54, P < 0.01), Group C vs. Group D+M+U (MD = 27.14, P < 0.01), Group D+U vs. Group M+U (MD = 7.56, P < 0.01), Group D+U vs. Group D+M+U (MD = 17.15, P < 0.01), Group M+U vs. Group D+M+U (MD = 9.60, P < 0.01). Compared with the control group, the levels of inflammatory factors in the ultrasound + schisandrin A (U+D) group and the ultrasound + microbubble group (U+M) showed a downward trend. The level of decrease was most pronounced in the ultrasound + schisandrin A + microbubble (U+M+D) group, as shown in Figure 6.

FIGURE 6.

In the control group, the tumor cells were closely arranged and the intercellular space was small, the nuclear showed atypia, the nuclei were large, the nuclear to cytoplasmic ratio was high, and mitotic figures were visible (red arrows), necrosis of tumor cells was occasionally observed (yellow arrow), the nuclei were pyknotic and hyperchromatic, there was a small amount of thin collagen fiber proliferation between the tumor cells (green arrow). There was no improvement in pathology for microbubble and ultrasound group. Compared with control group, in the ultrasound + schisandrin A group, the mitosis of tumor cell nuclei decreased (red arrows), there was a decrease in collagen fibers between tumor cells (green arrow), balloon-like swelling of tumor cells was observed (blue arrow). Compared with control group, in the ultrasound + microbubble group, tumor necrosis was increased (yellow arrow), the mitosis of tumor cell nuclei was decreased (red arrows), there was a decrease in collagen fibers between tumor cells (green arrow). Compared with control group, in the ultrasound + schisandrin A + microbubble group, tumor cell necrosis was significantly increased (yellow arrow), the mitosis of tumor cell nuclei was significantly decreased (red arrows), balloon-like swelling of tumor cells was significantly observed (blue arrow), there was a decrease in collagen fibers between tumor cells (green arrow), as shown in Figure 7.

FIGURE 7.

The results of immunohistochemistry showed that the control group (without treatment drugs) had a large staining area and strong staining intensity. There was no improvement in immunohistochemical result for microbubble and ultrasound group. Compared with the control group, the staining area of ultrasound + schisandrin A group was smaller and the staining intensity was weakened. Compared with the ultrasound + schisandrin A group, the staining area of the ultrasound + microbubble group and the ultrasound + schisandrin A microbubble group gradually became smaller, as shown in Figure 8. The above results showed that the expression of HIF-1α, VEGF and VEGFR-2 proteins in the tumor tissues of the ultrasound + schisandrin A group, the ultrasound + microbubble group and the ultrasound + schisandrin A microbubble group showed a weakening trend (P < 0.01), as shown in Figure 9.

FIGURE 8.

FIGURE 9.

Compared with the control group, the relative protein expression levels of p-PI3K, PI3K, p-Akt, AKT, p-mTOR, mTOR proteins in the ultrasound + schisandrin A group and the ultrasound + schisandrin A microbubble group were significantly decreased (P < 0.05), and the ultrasound + schisandrin A microbubble group had the most obvious effect. It was suggested that schisandrin A can inhibit the PI3K/AKT/mTOR signaling pathway in tumor tissues. After ultrasonograph-assisted microbubble destruction, the uptake of schisandrin A by tumor cells was further promoted, so the tumor inhibition effect was more obvious, as shown in Figure 10.

FIGURE 10.