Periplocin improves the sensitivity of oxaliplatin-resistant hepatocellular carcinoma cells by inhibiting M2 macrophage polarization

The aim of this research was to investigate the impact of periplocin (PPLN) on oxaliplatin (OXA) resistance in hepatocellular carcinoma (HCC) cells and offer insights for improving clinical treatment of HCC. The IC50 value of HCC cell lines against OXA was detected by the CCK-8 assay, and an OXA-resistant HepG2 cell line (HepG2/OXA) was constructed. THP-1 cells were induced into M1 or M2 macrophages, and M2 macrophage-conditioned medium (M2-CM) was prepared. M1 and M2 macrophage polarization were detected using RT-qPCR and flow cytometry. CCK-8, EdU staining, clone formation assay, flow cytometry, and western blotting were used to assess the proliferation and apoptosis of HepG2/OXA cells treated with PPLN and M2-CM. Additionally, a nude mouse subcutaneous graft tumor model was constructed. PPLN enhanced the sensitivity of HepG2/OXA cells to OXA, reduced their clone-forming ability, and promoted their apoptosis. Notably, PPLN hindered M0 macrophage polarization to M2 macrophages, while M1 polarization remained unaffected. The proliferation-inhibiting and apoptosis-promoting effects of OXA+PPLN on HepG2/OXA cells were significantly attenuated by the addition of M2-CM, suggesting that PPLN improves the OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization. Furthermore, PPLN inhibited M2 macrophage polarization and improved the OXA sensitivity of HepG2/OXA cells in vivo. In conclusion, PPLN inhibited the proliferation of HepG2/OXA cells, promoted their apoptosis, and inhibited M2 macrophage polarization both in vivo and in vitro, which in turn enhanced the OXA sensitivity of HepG2/OXA cells.


INTRODUCTION
Hepatocellular carcinoma (HCC) ranks as the fifth most prevalent malignant tumor globally and makes up 90% of all primary liver cancers (1,2).According to statistics, the five-year survival rate for HCC patients in Asian countries is a mere 18%, plummeting to 2% for those with advanced HCC (3,4).At early diagnosis, treatment options for HCC including surgical resection, liver transplantation, and local ablation (5,6).However, the majority of individuals with HCC come to the clinic when the tumor is already at an advanced stage and cannot be surgically removed.Chemotherapy is one of the mainstays of treatment for this group of patients, with platinum-based drugs having been commonly used in HCC treatment (7,8).
Cisplatin, a first-generation platinum-based drug, has become a major chemotherapeutic agent for the treatment of various solid tumors (9,10).The second-generation platinum-based anticancer drug carboplatin is structurally very similar to cisplatin, and its mechanism of action is also common to cisplatin, and it is also used for the treatment of various tumors (11).
Oxaliplatin (OXA) is a third-generation platinum-based anticancer drug and works in a similar way to other platinum-based drugs, the platinum atoms in OXA interact with DNA to block DNA replication and transcription (12,13).OXA has better therapeutic efficacy and higher tolerability than other platinum-based drugs, and OXA-based chemotherapy has been found to be beneficial for patients with advanced HCC (14,15).Although the therapeutic efficacy of platinum-based drugs for HCC has been recognized, HCC cells are highly susceptible to developing resistance to them, and the actual benefit to patients is very limited (16)(17)(18).Therefore, finding ways to reverse the resistance of HCC to platinum-based drugs has become a difficult and important part of HCC treatment.
Periplocae displays a range of pharmacological properties including cardiotonic, anti-tumor and anti-inflammatory, and its cardiotonic effect is mainly determined by cardiac glycosides (19).Periplocin (PPLN), a cardiac glycoside, is the main active ingredient of Periplocae, which inhibits the malignant advancement of various cancer types such as colorectal, gastric and pancreatic cancers (20)(21)(22).Bae et al. reported that PPLN hindered the proliferation of pancreatic cancer cells, leading to cell cycle arrest and apoptosis, and effectively increased the gemcitabine sensitivity of pancreatic cancer resistant cells (23).Notably, Lin et al. found that PPLN inhibited AKT/NF-κB pathway in HCC cells, which in turn inhibited cell proliferation, induced cycle arrest, and promoted apoptosis (24).However, the role of PPLN in HCC resistance to OXA is currently unclear.
Macrophages are major cells that exert immune functions.Several studies have shown the significant impact of tumor-associated macrophage M2 polarization on drug resistance in tumor cells (25,26).Therefore, we initiated this research by establishing a HepG2/OXA-resistant cell line and investigating the effects of PPLN and M2 macrophage polarization on HepG2/OXA cells.Next, it was explored whether PPLN affects M0 macrophage polarization toward M1 or M2, and further explored whether PPLN enhances the OXA sensitivity of HepG2/OXA cells by modulating M2 macrophage polarization.Finally, a nude mouse subcutaneous graft tumor model was constructed to investigate the effects of PPLN and OXA on tumors in nude mice in vivo.The aim of this research was to investigate the impact of PPLN on OXA resistance in HCC cells and offer insights for improving clinical treatment of HCC.

Establishment of the HepG2/OXA drug-resistant cells
HepG2 cells were seeded in 6-well plates and exposed to 5 μM OXA for 24 h after adherence, and cultured in DMEM complete medium (without OXA).When the cells recovered to a good state then add a larger concentration of OXA treatment according to the gradient concentration, the concentration of OXA added was in the following order : 10, 15, 20, 30, 40, 50, 70, 85, and 100 μM.After culturing the cells continuously for about 6 months in this method, a high concentration of drug-resistant cell line, HepG2/OXA, which was able to maintain a good growth state in medium containing 30.0 μM OXA, was obtained.When doing subsequent functional experiments, drug-resistant cells were first cultured under OXA-free conditions for more than a week to exclude the effect of OXA.

CCK-8 assay
HCC cells, M1 macrophages and M2 macrophages were seeded in 96-well cell culture plates (1.5×10 4 /well), and after attached to the wall, the original medium was changed to 200 μL of medium containing OXA (2.5, 5, 10, 20, 40, and 80 μM, HY-17371, MedChemExpress), PPLN (20,40,80,150, and 300 nM, HY-N1381, MedChemExpress) or M2-CM (1:1 ratio with DMEM medium).Following a 48-hour incubation period, 20 μL of CCK-8 reagent (C0038, Beyotime, Shanghai, China) was introduced into every well.After being incubated for 2 h at 37°C in an incubator protected from light, the OD450 value was measured utilizing a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).Based on the inhibition rate of cell viability by OXA and PPLN, CalcuSyn software 2.0 was used to calculate the combination index (CI) of the combined drugs as a way to determine whether the drugs were synergistic or not.

EdU staining
The EdU Cell Proliferation Detection Kit (C0071S, Beyotime) was used to identify the proliferation of HCC cells.Cells from different treatments were cultured for 48 h.After being rinsed twice with PBS, the cells were exposed to a 10 μM EdU culture solution for 1 h away from light.Following that, they were washed twice with PBS and exposed to 4% paraformaldehyde (Solarbio, Beijing, China) for 15 min.PBS including 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) was added to permeabilize for 10 min, Click reaction solution (Invitrogen, Carlsbad, CA, USA) was incubated for 30 min in a light-free environment, and then DAPI staining solution (Invitrogen) was about 10 min, after anti quenching sealing, under a fluorescence microscope observed and photographed subsequently.

Clone formation assay
Cells from each group in the logarithmic growth phase were taken, PBS washed and digested with 0.25% trypsin (Gibco) and blown into individual cells, which were then counted.Five hundred cells were placed in each well of a 6-well cell culture plate, respectively, and incubated for 14 d (37°C, 5% CO2).The culture was terminated when clonal cell clusters were visible to the naked eye.Aspirate the culture solution and rinse it twice with PBS.4% paraformaldehyde was added for fixation for 20 min.Then, the fixative was discarded and exposed to crystal violet (Sigma-Aldrich) for 15 min and then photographed for counting.

Flow cytometry
Cells from different treatments were cultured for 48 h, rinsed twice with PBS, and gently mixed by adding 500 μL of Binding Buffer.Then added Annexin-V-FITC (5 μL, MedChemExpress) and propidium iodide (5 μL, Beyotime) and mixed gently, strictly avoid light and incubated for 15 min.Flow-specific supersampling tubes were used to transfer the samples, and apoptosis was then identified through flow cytometry (BD FACSCaliburTM, BD biosciences, San Jose, CA, USA).

RT-qPCR
Total RNA was isolated from different sets of THP-1 cells using Trizol reagent (Invitrogen), and then reverse transcription was performed by adding AMV reverse transcriptase (Takara, Tokyo, Japan) to obtain cDNAs.Then, PCR amplification was carried out with TB Green® Premix Ex Taq™ II (TAKARA).Using β-actin as an internal reference, the relative expression of target genes was determined through the 2-ΔΔCt method.
The primer sequences used in this experiment are as follows: CD206: F:

Macrophage polarization marker assay
THP-1 cells were spread in 6-well plates and rinsed once with sterile PBS after different treatments.Cells were collected after centrifugation and resuspended in sterile PBS to make a single cell suspension (1.0×10 7 /mL).PE-labeled CD206 antibody (12-2061-82, Invitrogen) with FITC-labeled CD86 antibody (11-0862-82, Invitrogen) was added, mixed and incubated for 30 min away from light.Transferred to a flow cytometer for analysis, and the experimental results were analyzed and tallied using FlowJo software (v10.8,BD biosciences).

Western blot
RIPA lysate (P0013B, Beyotime) was utilized for lysing cells or tissues to extract proteins, and the BCA kit (P0012, Beyotime) was for assessing protein concentrations.The sample proteins were shifted to PVDF membranes (Invitrogen) after electrophoresis.After rinsing the membranes, they were placed at 4℃ for an overnight incubation with Bax primary antibody (MA5-14003, 1:100, Invitrogen) or Caspase 3 primary antibody (ab32351, 1:5000, Abcam, Cambridge, MA, USA).On the following day, after being rinsed thrice, the membranes were cultured with goat anti-rabbit secondary antibody IgG (31460, 1:10,000, Invitrogen).After exposure and development, the grayscale value of each protein band was assessed using Image J software, with β-actin (MA1-140, 1:5000, Invitrogen) serving as the internal reference.

Tumor-bearing assay in vivo
Balb/c female nude mice were purchased from Vitalriver (Beijing, China) at 4 to 6 weeks of age.The nude mice were kept in a stable environment at a temperature of 22°C and humidity ranging from 55% to 60%, following a 12-hour light and dark cycle.Nude mice were injected subcutaneously with 0.2 mL of HepG2/OXA cell suspension (5.0×10 6 cells/mice) and randomly divided into Control, OXA, PPLN, and OXA+PPLN groups.The administration was started on the 7th day after inoculation with 10 mg/kg OXA intraperitoneally in the OXA group, 15 mg/kg PPLN intraperitoneally in the PPLN group, 10 mg/kg OXA + 15 mg/kg PPLN intraperitoneally in the OXA + PPLN group, and an equal amount of substrate solution in the Control group.Administer the drug once daily for 7 days.The subcutaneous tumor dimensions were assessed on days 7, 14, 21, and 28 using a vernier caliper, and the mice were executed on d 28 after anaesthesia, the tumors were stripped and weighed.The study adhered to the guidelines approved by Guangzhou Meyers Biotechnology Co., LTD.
DNase-free proteinase K (20 μg/mL, ST532, Beyotime) was slowly added, followed by a 30-min incubation and three washes with PBS.TUNEL assay solution (C1086, Beyotime) was carefully added and left to incubate for 1.5 h away from light.Then added DAPI staining solution and incubated for 10 min, observed by fluorescence microscope and took photos.
On the following day, the secondary antibody was left to incubate at room temperature for 1 h before the color was developed in DAB solution (Solarbio), the reaction was terminated in distilled water, Mayer hematoxylin (Sigma-Aldrich) was used for re-staining, and the film was sealed with neutral dendrimer.Observed under an inverted microscope and photographed.

Immunofluorescence
Paraffin sections were deparaffinized with xylene, hydrated with gradient ethanol, and then antigenically repaired.Tissue sections were permeabilized with a drop of 0.3% Tritonx-100 (Sigma-Aldrich) for 10 min, and closed by adding bovine serum albumin for 2 h.Added CD206 antibody (1:500) or CD86 antibody (1:200) and incubated overnight at 4°C; on the next day, added FITC-labeled secondary anti-goat anti-rabbit IgG (1:10,000) and incubated for 1 h at 37°C away from light.Finally, DAPI stain was added and incubated for 10 min at room temperature away from light, and the development was observed with an inverted fluorescence microscope.

Ethical statement
The study adhered to the guidelines approved by Guangzhou Meyers Biotechnology Co., LTD.Laboratory animal center.

Statistical analysis
Every experiment was performed at least thrice, with the results were documented as the mean value ± corresponding standard deviation.SPSS 26.0 software (IBM SPSS Statistics 26) was utilized to process and analyze the data statistically.Student's t-test was utilized to evaluate the distinctions between the two groups.ANOVA was applied to make comparisons between sub-multiple groups.Prism software (Graphpad 9.0) was utilized for plotting.*P<0.05signifying that there was a significant difference.

Construction and validation of OXA-resistant HCC cell lines
Previous studies have shown that Huh-7, HepG2, and MHCC-97H cells are commonly used HCC cell lines for studying OXA resistance (27,28).The IC50 of different HCC cell lines against OXA was detected by CCK-8 assay, and the findings revealed that the IC50 values of Huh-7, HepG2 and MHCC-97H cells were 13.80, 8.58, and 25.16 μM, respectively (Figure 1A).This showed that HepG2 cells are the most sensitive to OXA, so we selected HepG2 cells to construct an OXA-resistant cell line (HepG2/OXA).The IC50 value of HepG2/OXA cells was 33.07 μM, which was approximately four times higher than the IC50 value of HepG2 cells (8.45 μM), suggesting that the HepG2/OXA-resistant cell line was constructed successfully (Figure 1B).Since the IC50 value of Huh-7 cells was 8.45 μM, we treated HepG2 cells and HepG2/OXA cells with 10 μM OXA in subsequent experiments.EdU staining results indicated a noticeable decrease in the amount of EdU positivity of HepG2 cells compared to HepG2/OXA cells, indicating that the inhibition of OXA on the proliferation of HepG2 cells was notably greater compared to HepG2/OXA cells (Figures 1C-1D).The clone formation experiments demonstrated a marked increase in the amount of clones in HepG2/OXA cells in comparison to HepG2 cells after OXA treatment (Figures 1E-1F).Additionally, flow cytometry indicated a notable increase in apoptosis rate in HepG2/OXA cells compared to HepG2 cells following OXA treatment (Figures 1G-1H).
These results further confirmed the successful construction of HepG2/OXA-resistant cells.

PPLN enhances OXA sensitivity in HepG2/OXA cells
The activity of HepG2/OXA cells was assessed using the CCK-8 assay to determine the impact of PPLN, revealing a marked reduction in cell activity at 80, 150, and 300 nM concentrations of PPLN, and the effect was concentration-dependent (Figure 2A).In the subsequent experiments, we chose 80 nM PPLN to treat the cells.Additionally, the sensitivity of HepG2/OXA cells to OXA was enhanced by PPLN treatment, as evidenced by a decrease in the IC50 value to 17.51 μM (Figure 2B).Notably, the CI was less than 1 after 48 h of OXA and PPLN co-treatment, suggesting a synergistic inhibitory effect of OXA and PPLN on HepG2/OXA cell proliferation (Figure 2C).EdU staining results showed that either OXA or PPLN treatment significantly reduced the number of EdU positivity in HepG2/OXA cells, while OXA+PPLN treatment was significantly more effective than the single-treatment group (Figures 2D and 2F).The results of clone formation experiments showed that either OXA or PPLN treatment significantly reduced the number of clones in HepG2/OXA cells, while OXA+PPLN treatment further reduced the number of cloned cells (Figures 2E and 2G).Flow cytometry results showed that OXA or PPLN treatment significantly reduced the apoptosis rate of HepG2/OXA cells, while OXA+PPLN treatment led to a further reduction in apoptosis rate (Figures 2H-2I).Not only that, Western blot assay showed that the levels of apoptosis-related proteins Bax and Caspase 3 were both markedly elevated in HepG2/OXA cells after OXA or PPLN treatments, and OXA+PPLN treatments led to a further elevation of their expression (Figures 2J-2K).The findings indicated that PPLN enhances the effectiveness of OXA and heightens the OXA sensitivity of HepG2/OXA cells.

M2 macrophages increase OXA resistance in HepG2/OXA cells
To examine the potential effect of M2 macrophages on the sensitivity of HepG2/OXA cells to OXA, we supplemented the cell culture system with M2-CM and exposed it to 10 μM OXA for 48 h.Following the addition of M2-CM, the IC50 value of HepG2/OXA cells against OXA increased to 42.23 μM, as revealed by the results of the CCK-8 assay (Figure 3A).By EdU staining, we observed that OXA treatment significantly reduced the number of EdU positivity in HepG2/OXA cells, whereas the inhibitory influence of OXA on the proliferation of HepG2/OXA cells was attenuated by the addition of M2-CM to the culture system (Figures 3B and 3E).In addition, the amount of clones in HepG2/OXA cells was notably diminished with OXA treatment, but the suppressive impact of OXA on the clone formation ability of HepG2/OXA cells was alleviated by the addition of M2-CM (Figures 3C and 3F).
We examined the apoptosis rate of HepG2/OXA cells by flow cytometry and showed that OXA+M2-CM treatment attenuated apoptosis in HepG2/OXA cells compared to OXA treatment alone (Figures 3D and 3G).Not only that, Western blot assay showed that the levels of Bax and Caspase 3 in HepG2/OXA cells was significantly elevated after OXA treatment, whereas the effect of OXA was weakened by the addition of M2-CM (Figures 3H-3I).These findings indicated that M2 macrophages attenuated the impact of OXA and increase OXA resistance in HepG2/OXA cells.

PPLN inhibits M2 macrophage polarization
CCK-8 assay results showed that the effect of PPLN treatment on M1 macrophage and M2 macrophage viability was not significant (Figures 4A-4B).Next, we examined the levels of markers for M2 and M1 macrophage polarization following PPLN treatment to explore its influence on macrophage polarization.The results of RT-qPCR indicated a significant increase in the expression of M2 macrophage polarization markers CD206, IL-10, Arg1, and Fizz1 following treatment with IL-4+IL-13, whereas PPLN decreased the levels of these markers, suggesting that PPLN inhibits M2 macrophage polarization (Figures 4C-4F).Not only that, the levels of M1 macrophage polarization markers CD86, iNOS, IL-6 and TNF-α were significantly elevated after LPS+IFN-γ treatment, the presence of PPLN did not result in any noticeable changes in the expression of these markers, indicating that PPLN does not impact the polarization of M0 macrophages into M1 (Figures 4G-4J).Flow cytometry results similarly showed that PPLN treatment significantly reduced the number of CD206-positive cells, whereas there was no significant change in the number of CD86-positive cells, further confirming that PPLN inhibited M2 macrophage polarization but did not affect M1 macrophage polarization (Figures 4K-4L).

PPLN enhances OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization
To determine whether PPLN enhances OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization, we added PPLN and M2-CM to the HepG2/OXA cell culture system and detected cell viability utilizing CCK-8 assay.After PPLN treatment, the IC50 value of HepG2/OXA cells against OXA decreased to 17.16 μM, whereas the IC50 value was elevated to 24.66 μM in the simultaneous presence of PPLN and M2-CM, which suggested that M2-CM attenuated the enhancing impact of PPLN on the chemosensitivity of HepG2/OXA cells (Figure 5A).By EdU staining, we observed a significant attenuation of EdU fluorescence intensity and a notable decline in the number of EdU-positive cells in OXA+PPLN-treated HepG2/OXA cells, whereas the addition of M2-CM attenuated the effect of OXA+PPLN (Figures 5B and 5E).Not only that, we found that OXA+PPLN treatment reduced the clone formation ability of HepG2/OXA cells, which was reversed by the addition of M2-CM (Figures 5C and 5F).Flow cytometry results showed that OXA+PPLN treatment led to a marked increase in the apoptosis rate of HepG2/OXA cells, whereas the addition of M2-CM attenuated the apoptosis-promoting effect of OXA+PPLN (Figures 5D and 5G).In addition, the levels of Bax and Caspase 3 were notably elevated in HepG2/OXA cells after OXA+PPLN treatment, whereas the addition of M2-CM reduced the effect of OXA+PPLN (Figures 5H-5I).The above results indicated that M2 macrophage polarization attenuated the effect of OXA+PPLN, suggesting that PPLN improved the sensitivity of HepG2/OXA cells to OXA by hindering M2 macrophage polarization.

PPLN enhances OXA sensitivity in HepG2/OXA cells in vivo
Finally, a nude mice xenograft tumor model was constructed according to the process shown in Figure 6A to explore the impacts of OXA and PPLN on tumor growth in vivo.The results showed that injection of either OXA or PPLN significantly reduced tumor volume and weight in nude mice in vivo, the combined OXA+PPLN injection resulted in more pronounced inhibition of tumor growth compared to the individual treatment (Figure 6B-6D).In addition, Tunel staining revealed a marked rise in the number of Tunel-positive cells in the tumor tissues following the injection of OXA or PPLN, which was further elevated after injection of OXA+PPLN (Figure 6E).We used immunohistochemistry to assess the expression of M2 macrophage and M1 macrophage polarization markers in HCC tissues, and the results revealed that M2 macrophage polarization markers CD206 and Arg1 were notably down-regulated by injection of OXA or PPLN, and was further reduced by injection of OXA+PPLN (Figure 6F-6H).In contrast, injection of OXA, PPLN, or OXA+PPLN had no significant effect on the expression of the M1 macrophage markers CD86 and iNOS (Figure 6I-6J).In addition, immunofluorescence results also showed that injection of OXA or PPLN significantly reduced the number of CD206-positive cells, and OXA+PPLN treatment further reduced it, but none of the above treatments affected the number of CD86-positive cells (Figure 6K-6M).These results further indicated that PPLN hindered M2 macrophage polarization in vivo, consequently increasing the sensitivity of HepG2/OXA cells to OXA.

DISCUSSION
Continuous OXA chemotherapy is prone to lead to drug resistance in HCC cells, which severely limits its efficacy in clinical applications (29,30), exploring safe drugs for chemosensitization has positive implications for the treatment of HCC patients.It has been reported that drug resistance can be categorized into acquired and natural resistance depending on the reason for its occurrence, and that acquired resistance can be caused by culturing cells by exposing them to chemotherapeutic drugs on a continuous basis (31,32).
Other studies have indicated that several biological processes including drug efflux pumps, DNA damage repair, apoptosis, hypoxia, and epigenetic modifications are associated with OXA resistance (33).Therefore, establishing OXA-resistant cell lines in vitro, focusing on enhancing the OXA sensitivity of HCC cells, and exploring the possible mechanisms will help to solve the challenge of limited efficacy of OXA.Through a gradual increase in drug concentration, we developed a HepG2/OXA resistant cell line in this research, and the proliferation-inhibiting and apoptosis-promoting effects of OXA on HepG2 cells were significantly stronger than those on HepG2/OXA cells, which further proved that the construction of HepG2/OXA drug-resistant cell line was successful.
In recent years, the role of herbal extracts in inhibiting malignant progression of tumors has attracted much attention because of their safety, low toxicity and side effects, multi-pathway and multi-target characteristics (34,35).As the main extract of Periplocae, PPLN has strong antitumor activity.Zhao et al. showed that PPLN promoted apoptosis in gastric cancer cells through ERK1/2-EGR1 pathway, and the combination of PPLN and TRAIL resulted in a greater reduction in cell viability compared to individual treatments, suggesting that PPLN increased the TRAIL sensitivity of gastric cancer cells (36).We found that PPLN dose-dependently decreased the activity of HepG2/OXA cells, lowered the IC50 value of HepG2/OXA cells for OXA, and enhanced the effect of OXA in hindering proliferation and inducing apoptosis both in vivo and in vitro, implying that it could improve the OXA sensitivity of HCC cells, which is similar to the previous findings.
Macrophages alter their phenotype to restore homeostasis in the body to enhance their ability to cope with changes in the surroundings, a phenomenon referred to as macrophage polarization (37).Diversity and plasticity are classical features of macrophages, which differentiate into two classical phenotypes: the M1 and M2, with the M1 macrophage acting as a classically activated macrophage with pro-inflammatory properties and the M2 macrophage acting as a selectively activated macrophage with anti-inflammatory properties (38).Exposure to pathogen-associated molecules including IFN-γ, TNF-α, or LPS causes macrophages to polarize to the M1 type and secrete abundant quantities of pro-inflammatory factors, which dominate the inflammatory microenvironment unfavorable to tissue recovery and participate in anti-tumor immunity (39,40).Whereas exposure to IL-4, IL-10, glucocorticoids, TGFβ, or immune complexes triggered M2 macrophage polarization (41).
M2 macrophages release a variety of chemokines and anti-inflammatory substances to avoid excessive damage to the body and promote wound healing (42).M1 macrophages highly express CD80, iNOS, CD86, IL-6, and TNF-α, compared with elevated expression of Fizz1, Arg-1, CD206, IL-10, CCL17, and CCL22 in M2 macrophages (43)(44)(45).Notably, our results showed that PPLN reduced the levels of M2 macrophage polarization markers both in vivo and in vitro, with no notable impact on the expression of M1 polarization markers, suggesting that PPLN inhibits M2 macrophage polarization.
Evidence is mounting to support the idea that M2 macrophage polarization is a significant factor in fueling the progression of malignant tumors (46,47).Chen et al. reported that CHI3L1 protein released by M2 macrophages promoted metastasis of gastric and breast cancer cells (48).Yu et al. indicated that M2 macrophage-derived exosomes circ 0008253 reduced the impact of OXA on gastric cancer cells and decreased apoptosis rate, suggesting that M2 macrophage-derived exosomes enhance OXA resistance in gastric cancer cells (49).
Not only that, Qu et al. found that M2 macrophage polarization increased cisplatin resistance in gastric cancer cells (50).For this research, we prompted M0 macrophages polarization into M2 macrophages to prepare M2-CM for mimicking tumor-associated M2 macrophage infiltration in vitro.We found that M2 macrophages attenuated the effect of OXA and increased the resistance of HepG2/OXA cells to OXA, similar to previous findings.Furthermore, To determine whether PPLN enhances the sensitivity of HepG2/OXA cells to OXA by inhibiting M2 macrophage polarization, we added PPLN and/or M2-CM to the HepG2/OXA cell culture system.We found that the addition of M2-CM weakened the inhibitory effect of OXA PPLN on cell proliferation and the promoting effect on cell apoptosis, attenuated the chemosensitizing effect of PPLN, suggesting that PPLN improves the OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization.

CONCLUSION
PPLN enhanced the impact of OXA, decreased the viability and proliferation of HepG2/OXA cells, and promoted their apoptosis.In addition, M2 macrophage polarization increased the OXA resistance of HepG2/OXA cells, whereas PPLN inhibited M2 macrophage polarization, suggesting that PPLN enhanced the OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization.This study elucidated the action mechanism of PPLN in enhancing the chemosensitivity of HCC cells by in vitro and in vivo experiments, which provides new references for the clinical application of PPLN, the treatment of chemotherapy-resistant patients and the improvement of their prognosis.However, there are still some shortcomings, and further exploration of the signaling pathways through which PPLN enhances the chemosensitivity of HCC cells is needed subsequently.

Figure 1 .Figure 2 .
Figure 1.Construction and validation of OXA-resistant HCC cell lines.(A-B) The IC50 of Huh-7, HepG2, MHCC-97H cells and HepG2/OXA cells against OXA was assessed by CCK-8 assay.(C-D) EDU staining was utilized to determine the influence of OXA (10 μM) on HepG2 and HepG2/OXA cell proliferation.(E-F) Clone formation assay was utilized to detect the impact of OXA (10 μM) on clone formation in HepG2 and HepG2/OXA cells.(G-H) The apoptosis rate of HepG2 and HepG2/OXA cells after OXA (10 μM) treatment was determined utilizing Flow cytometry

Figure 3 .
Figure 3. M2 macrophages increase OXA resistance in HepG2/OXA cells.(A) The IC50 of HepG2/OXA cells against OXA in the presence or absence of M2-CM were assessed by CCK-8 assay.(B and E) EDU staining was utilized to detect the effect of OXA, M2-CM or OXA+M2-CM co-treatment on the proliferation of HepG2/OXA cells.(C and F) Clone formation assay was utilized to examine the effects of OXA, M2-CM, or OXA+M2-CM co-treatment on clone formation in HepG2/OXA cells.(D and G) The apoptosis rate of HepG2/OXA cells after OXA, M2-CM or OXA+M2-CM co-treatment was quantified utilizing Flow cytometry.(H-I) Western blot was utilized to quantify Bax and Caspase 3 levels in HepG2/OXA cells after OXA, M2-CM or OXA+M2-CM co-treatment.

Figure 6 .
Figure 6.PPLN enhances OXA sensitivity in HepG2/OXA cells in vivo.(A) The process of constructing nude mice subcutaneous graft tumor model.(B) The effect of tumor growth after subcutaneous injection of OXA (10 mg/kg), PPLN (15 mg/kg), or OXA+PPLN compared to control (n=5) (day 28).(C) Measurement of the volume of subcutaneous grafted tumors in nude mice at 7, 14, 21 and 28 d after different treatments.(D) Measurement of tumor weights in nude mice after different treatments (d 28).(E) Tunel staining was utilized to examine apoptosis in tumor tissues of nude mice.(F-J) The levels of M2 macrophage markers (CD206 and Arg1) and M1 macrophage markers (CD86 and iNOS) in tumor tissues was assessed by immunohistochemistry. (K-M) Immunofluorescence detected M2 macrophage marker CD206 and M1 macrophage marker CD86 expression in tumor tissues.

Figure 7 .
Figure 7. PPLN inhibits M2 macrophage polarization, suppresses proliferation and promotes apoptosis of OXA-resistant HCC cells both in vivo and in vitro, which in turn enhances the sensitivity of drug-resistant cells to OXA.