Lobaplatin induces apoptosis in T24 and 5637 bladder cancer cells by regulating Bcl-2 and Bax expression and inhibiting the PI3K/Akt signaling pathway

Introduction

Bladder cancer (BC) is one of the most common cancers in the world and has a higher morbidity and mortality rate (1). According to prognostic features, BC is divided into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) (1-3). Clinically, 70% of patients are first diagnosed with NMIBC. However, after receiving transurethral resection, high-risk patients will relapse and develop MIBC (4). Therefore, it is of great significance to develop or identify safe and effective new drugs for BC treatment.

Lobaplatin (LBP), a third-generation platinum-based drug only available in the Chinese market, has been approved for treating metastatic BC, chronic granulocytic leukemia, and small cell lung cancer (5), for which surgical removal is not an option. In addition, some studies have confirmed that LBP is effective in treating other cancer types, including cervical cancer, triple-negative breast cancer, primary hepatocellular carcinoma, and T4 gastric cancer (6-9). Compared to other platinum-based chemotherapy drugs on the market, such as cisplatin (DDP), LBP is less toxic to the kidney, nerves and ear and bypasses the DDP resistance of cancer cells (10). Considering the similar antitumor mechanisms of LBP and DDP—both can cause DNA damage by generating DNA adducts, thereby inhibiting cell proliferation and inducing apoptosis—in the present study, for the first time, we explored whether LBP has a similar effect to DDP in treating BC while taking into account multiple pathways in the tumor cells related to the occurrence of BC, such as the overactivation of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway in MIBC (11). In this study, we further investigated whether the potential efficacy of LBP in treating BC is related to its ability to regulate B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) expression and to inhibit the PI3K/Akt pathway in the hope of laying an experimental foundation for LBP application in the clinical treatment of BC. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-23-376/rc).

Methods

Materials

T24 human BC cells (SCSP-536) and the human BC cell line 5637 (TCHu 1) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Male BALB/c nude mice (BALB/c nude mice, little individual variation, commonly used in xenograft tumor models) 35–48 days old (grade: specific pathogen free) were purchased from Charles River Laboratories [license key: SCXK (Jing) 2016-0006] (Beijing, China). LBP lyophilized powder for injection was provided by Hainan Changan International Pharmaceutical Co., Ltd. (Hainan, China). Fetal bovine serum (FBS) was provided by HyClone (Logan, Utah, United States). Roswell Park Memorial Institute-1640 medium was provided by GE Healthcare Life Sciences (Pittsburgh, Pennsylvania, United States). Cell counting kit-8 (CCK-8) reagents were purchased from Dojindo Molecular Technologies (Tokyo, Japan). The FITC annexin V cell apoptosis assay kit was provided by BD Biosciences (Bergen, New Jersey, United States), and trypsin was purchased from Beijing Solarbio Life Sciences (Beijing, China).

Antibodies

Bcl-2 (D17C4) rabbit monoclonal antibody (mAb), Bax (D2E11) rabbit mAb, caspase-3 rabbit mAb, cleaved caspase-3 (Asp175) antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11) XP rabbit mAb, β-Tubulin (9F3) rabbit mAb, PI3K p110a (C73F8) rabbit mAb, Akt (pan) (11E7) rabbit mAb, phospho-Akt (Ser473) (D9e) XP rabbit mAb and anti-rabbit IgG, Horseradish Peroxidase (HRP)-linked antibody was purchased from Cell Signalling Technology (Boston, Massachusetts, USA) were obtained for these studies. Fluorescent secondary antibodies were purchased from LI-COR Biosciences (Lincoln, Nebraska, United States).

Cell culture

T24 and 5637 cells were cultured separately in RPMI-1640 medium containing 1% penicillin-streptomycin and 10% FBS (complete medium). The complete medium was used and replaced every 2 days until 75–90% of cells were attached in a petri dish at 37 ℃ in an incubator with 5% CO₂. The cells that reached the required number were used in experimental studies. The cells were subcultured at a 1:3 ratio.

CCK-8 assay for assessing cell viability

T24 and 5637 cells in the logarithmic growth phase were selected under a microscope. The original medium was discarded, and the cells were washed with phosphate buffered saline (PBS). Complete medium was used to resuspend the cell pellets after trypsin digestion. A total of 1×10⁴ cells were counted in 100 µL per well and transferred to a 96-well plate. After the addition of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt, the absorbance at 450 nm was measured by an Enzyme-labeled instrument. The experiment was repeated at least 3 times, and 3 experimental wells were set in each group. The optical density (OD) was measured at a wavelength of 450 nm:

Cellviability(%)=(ODexperimentalgroup−ODblankgroup)(ODcontrolgroup−ODblankgroup)×100%[1]

Wound healing assay

A wound healing assay (12) was employed to determine the inhibitory effect of LBP on the migration of the two BC cell lines, namely, the T24 and 5637 lines. T24 and 5637 cells were seeded in a six-well culture plate (5×10⁵ cells per well) until the cell confluency reached 80%. Then, the medium was removed, and a “wound” was created. After washing with PBS twice, we added culture medium containing LBP at different concentrations (0, 1, 2, 4, and 8 µg/mL) and captured images of the wound at the time of wounding (T₀). After 24 hours (T₂₄), we took photos of the same area and measured the wound size at T₀ and T₂₄, based on which the rate of cell migration was calculated to estimate the migration capacity of cells, with the formula shown below. The experiment was repeated at least 3 times.

Cellmigrationrate(%)woundhealingrate(%)=(woundsizeatT0−woundsizeatT24)woundsizeatT0×100%[2]

Apoptosis detection with annexin V-FITC/PI staining

T24 and 5637 cells were treated with 0 and 8 µg/mL LBP for 24 hours. The adherent cells on the bottom were made into a cell suspension after trypsin digestion. After the suspension was centrifuged for 7 min at 1.0×10³ r·min⁻¹, PBS was used to wash the cells three times. According to the instructions of the Annexin V-FITC cell apoptosis kit. At room temperature, the cells were cultured in the dark for 15 min and analyzed with a flow cytometer. The experiment was repeated at least 3 times.

Western blot analysis of Bcl-2, Bax, caspase-3, cleaved caspase-3, PI3K, Akt and p-Akt protein expression

Whole-cell proteins were extracted using RIPA lysate containing 1 mM PMSF, and the same amount of protein (50 µg) was separated via electrophoresis and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. Tris Buffered Saline with Tween (TBS-T) solution containing 5% skimmed milk was used to block the PVDF membrane for 90 min at room temperature. Afterward, the blocked PVDF membrane was incubated with primary antibodies overnight at 4 ℃. The excess primary antibody was washed off, and then, the membrane was incubated with goat anti-rabbit IgG (1:1,000) at room temperature for 1.5 hours. Eventually, the membrane was evaluated using an Odyssey dual-color infrared laser imaging system, which was developed by LI-COR, and Image J for greyscale analysis [The infrared laser produces two wavelengths of laser light (680 and 780 nm) which excite the infrared fluorescent dye to produce emitted light at 720 and 820 nm respectively, which is then simultaneously detected by two highly sensitive avalanche photodiodes to detect the signal intensity].

Nude mouse xenograft assay

Eight male BALB/c nude mice from each experimental group were selected randomly, with two groups containing a total of 16 mice. Serum-free medium was used to dilute T24 and 5637 cells to 2×10⁷ cells/mL. Then, 200 µL of cell suspension was injected subcutaneously into the backs of the nude mice, generating a solid tumor. When the solid tumor grew to 200 mm³, the nude mice were randomly split into the control group and the treatment group, and the latter group received an intravenous injection of 25 mg·kg⁻¹ LBP. Then, we measured the weight of the nude mice and the tumor size every 3 days and extracted the tumor tissue after 21 days for weighing and cryopreservation. Mice who died were excluded from the experiment. Tumor size was calculated as follows:

Tumorsize=(L×W2)/2[3]

(L refers to the length of the tumor tissue and W the width).

Immunohistochemical test

The tumor tissues were made into paraffin sections and deparaffinized with ethanol and xylene at different concentrations. Then, the deparaffinized sections were boiled in 0.01 M citrate buffer, incubated in 3% H₂O₂ for 10 min to reduce the activity of endogenous peroxidase, blocked in 5% nonfat milk for 60 min, and incubated with antibodies against Bax, Bcl-2, and cleaved caspase-3 at 4 ℃ overnight. The next day, we incubated the sections with HRP-linked antibody for 60 min, followed by staining through incubation in 3,3-diaminobenzidine (DAB) substrate for 10 min. Then, we took photos of the obtained sections with an optical microscope and processed the photos. Semi-quantitative analysis of the staining results: the intensity of the staining is combined with the percentage of positive cytology to give a composite score, with positive cells marked by a yellowish to brownish stain in the cytoplasm of the tissue section. The intensity of staining is based on the staining characteristics of the majority of cells (shades of staining in contrast to background staining): 0 points for no staining, 1 point for pale yellow, 2 points for brown, and 3 points for tan. The percentage of positive cells is the average number of positive cells over 5 fields of view (100 such cells counted in a 400× high magnification field) (13).

Animal experimental ethical inspection

The in vivo experiment was performed according to the Guide for the Care and Use of Laboratory Animals of Jilin University and was approved by the ethics committee of Jilin University School of Pharmaceutical Science (No. 20190055). A protocol was prepared before the study without registration.

Statistical and analytical methods

SPSS 24.0 software, developed by International Business Machines Corporation (IBM), was selected to process the data, and ImageJ software was used to quantitatively analyze the grayscale values of the protein bands and the cell scratch area. According to the experimental groups, the data from two groups were compared with unpaired t-tests, and multiple groups were compared via analysis of variance (ANOVA). A value of P<0.05 was regarded as statistically significant.

Estimation was carried out by means of the degrees of freedom (E) of the analysis of variance. The range of E should be between 10 and 20. If the calculated E is less than 10, it means that the number of animals in each group should be increased as a way to increase the likelihood of producing significance-level results; when the calculated E is greater than 20, it means that the number of animals estimated is too high and the likelihood of producing significance level results does not continue to increase, resulting in a waste of experimental animals; thus, the number of animals in each group should be reduced. E is calculated by the following formula:

E=sumofexperimentalanimalsineachgroup−numberofgroups[4]

The RAND function in Excel was used to create random arrays to group mice. Experiments were conducted by random number assignment results to reduce potential confounding factors.

Results

Inhibitory effects of LBP on T24 and 5637 cell viability

LBP impacts on the growth of BC cells were observed through the detection of cell viability. As shown in Figure 1, after exposure to LBP for 24 hours (LBP concentration ≥1 µg/mL), the viability of T24 and 5637 cells was obviously inhibited compared to that in the control group, and after 48 and 72 hours (LBP concentration ≥0.5 µg/mL), the viability of the cells was significantly inhibited, which suggests that the inhibition of cell viability is dependent on the dose of LBP. Since the effects of the treatment over different lengths of time were all significant, we chose 24 hours for the subsequent experiment (P<0.05). Under this condition, the half-maximal inhibitory concentration (IC50) of LBP in T24 cells was 11.623 µg/mL, while the IC50 in 5637 cells was 9.611 µg/mL. Based on the IC50, 8 µg/mL was chosen for the subsequent experiment.

Figure 1 Effects of different concentrations of LBP on T24 and 5637 cell viability after 24, 48 and 72 hours. Compared with the control group, *, P<0.05, **, P<0.01. LBP, lobaplatin.

The effects of LBP on the migration rate of human BC cells

The duration of treatment was set as 24 hours according to the experimental results LBP at 0 µg/mL was set as the control group, and LBP at 1, 2, 4, and 8 µg/mL was set as the treatment group. As displayed in Figure 2, the migration rate of T24 cells in the control group was 60.49%±3.38%; the rate in the LBP 1 µg/mL group (51.67%±6.30%) was not significantly different (P>0.05); the rates at LBP 2 µg/mL (36.78%±6.67%), 4 µg/mL (31.43%±3.12%) and 8 µg/mL (26.85%±3.94%) were significantly different (P<0.01). The migration rate of 5637 cells in the control group was 58.37%±4.68%, the migration rate at LBP 1 µg/mL (50.45%±4.01%) was not significantly different (P>0.05); the rates obtained at LBP 2 µg/mL (41.77%±10.54%), 4 µg/mL (36.94%±1.78%) and 8 µg/mL (31.83%±2.51%) were significantly different from that in the control group (P<0.05).

Figure 2 Effect of LBP on cell (T24 and 5637) migration ability. Compared with the control group, *, P<0.05, **, P<0.01. LBP, lobaplatin. 40×, microscopic observation.

The effects of LBP on BC cell apoptosis

According to the experimental results given above, we set the duration of treatment as 24 hours, with LBP at 0 µg/mL as the control group and LBP at 8 µg/mL as the treatment group. Annexin V-FITC/PI staining was employed to analyze the level of cell apoptosis. According to the results presented in Figure 3, the apoptosis of T24 cells in the treatment group (31.25%±1.20%) was greater than that in the control group (6.25%±0.64%); the apoptosis of 5637 cells in the treatment group (14.3%±2.24%) was greater than that in the control group (2.5%±0.78%). All the differences were significant (P<0.05).

Figure 3 Flow cytometry was used to detect the effect of LBP on the proportion of apoptotic cells. *, P<0.05; **, P<0.01, compared with the control group (the Y-axis is PI and the X-axis is annexin-V FITC). LBP, lobaplatin; PI, propidium iodide; FITC, fluorescein isothiocyanate isomer I.

The effects of LBP on the expression of apoptosis-related proteins and PI3K/Akt pathway proteins

Western blotting was used to analyze the expression levels of apoptosis-related proteins (Bax, Bcl-2, caspase-3 and cleaved caspase-3) and proteins in the PI3K/Akt pathway in T24 and 5637 cells before and after LBP treatment. As displayed in Figure 4, compared with that in the control group, after 24 hours of exposure to LBP, the expression of Bcl-2 protein decreased in both cell lines and that of Bax protein increased; the Bcl-2/Bax ratio decreased; the activation of caspase-3 and cleaved caspase-3 was enhanced; the expression of PI3K, AKT and p-Akt in T24 cells decreased and that of PI3K and p-Akt in 5637 cells decreased (P<0.05); and no significant changes were observed in Akt (P>0.05).

Figure 4 LBP regulates bladder cancer cell growth through the PI3K/AKT signalling pathway. (A-C) The effect of LBP on the expression of apoptosis-related proteins and PI3K/Akt pathway proteins in T24 cells. (D-F) The effect of LBP on apoptosis-related proteins and PI3K/Akt pathway expression in 5637 cells. *, P<0.05; **, P<0.01, compared with the control group. LBP, lobaplatin; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

The effects of LBP on the growth of tumor xenografts

A nude mouse model with T24 and 5637 tumor xenografts was constructed to evaluate the effects of LBP in inhibiting BC. Compared with that in the control group, the tumor size in nude mice treated with LBP (25 mg·kg⁻¹) was considerably smaller (Figure 5A,5B). There was no significant change in the body weight of nude mice in the treatment and model groups (Figure 5C,5D). The tumors in the treatment group grew slowly and tumor volumes were significantly less (Figure 5E,5F).

Figure 5 In vivo efficacy of LBP treatment in nude mouse models of T24 and 5637 tumour xenografts. (A,B) Photo of the tumors removed from mice in the two experimental groups after three weeks. (C) Body weight curves of T24 tumor xenografts in the nude mice in the two groups. (D) Body weight of 5637 tumor xenografts in the nude mice in the two groups. (E) Comparison of the tumor volume of T24 tumor xenografts in the two groups of nude mice. (F) Comparison of the tumor volume of 5637 tumor xenografts in the two groups of nude mice. Scale bars, 10 mm. *, P<0.05; **, P<0.01. LBP, lobaplatin.

The effects of LBP on apoptosis-related proteins in tumor xenografts

Immunohistochemistry was used to detect the expression level of apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase-3) in the tumor tissue. The staining results of tumor xenografts induced by T24 and 5637 BC cells are presented in Figure 6. Compared with that in the control group, the expression of Bax increased in mice treated with LBP (25 mg·kg⁻¹), that of Bcl-2 protein declined, the Bcl-2/Bax ratio decreased significantly (P<0.05), and cleaved caspase-3 expression in the tissue was increased, indicating activation.

Figure 6 IHC staining images of the bladder tumor tissue in mice and analyses. Effect of LBP on the expression of Bax, Bcl-2 and cleaved caspase-3 in tumour sections using immunohistochemical staining of tissue wax blocks (×200). (A,B) The effect of LBP on the expression of apoptosis-related proteins in T24 metastatic tumors; (C,D) the effect of LBP on the expression of apoptosis-related proteins in 5637 metastatic tumors. *, P<0.05; **, P<0.01. LBP, lobaplatin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2.

Discussion

The growth of bladder tumors is determined by the state of balance between the proliferation and apoptosis of tumor cells. Chemotherapy is a treatment that inhibits tumor growth by inducing cell cycle arrest and apoptosis. Shortly after its development, the third-generation platinum-based drug LBP [1,2-diamminomethyl-cyclobutane-platinum (II)-lactate] was found to be effective in inhibiting the growth of multiple types of tumors in mice (14-16). Later, further research confirmed its efficacy in inducing the apoptosis of many types of cancer cells, including BGC-823 gastric cancer cells (through the ROS-mitochondrial pathway) (17), nasopharyngeal cancer cells (by regulating cIAP1/2, ribosomes and ROS) (18), and non-small cell lung cancer cells (by acting on the p53/ROS/p38 MAPK pathway) (15).

In this study, for the first time, we explored whether LBP is effective in inhibiting the proliferation of BC cells. This study focused on T24 as the MIBC cell line and 5637 as the NMIBC cell line because of their different chemosensitivity levels and their wide application in BC research (19). The experimental results confirmed that LBP reduces the viability of T24 and 5637 cells and inhibits their migration in a dose-dependent manner. Moreover, when the concentration of LBP reached the IC50 value, LBP was able to induce apoptosis in the two types of BC cells. At the same time, necrotic and early apoptotic cells were further increased after drug administration. This dose-dependent characteristic of LBP was also confirmed in the study of Yu et al. in rectal cancer cells (20). In addition, cell migration ability is a key indicator to evaluate the ability of cancer cells to spread (21,22), and LBP was shown to have an inhibitory effect on the migration of the prostate cancer cell lines DU145 and PC3 (23). This inhibitory effect of LBP on cell migration was confirmed in T24 and 5637 cells in our study, providing some degree of data support for the ability of LBP to inhibit the migration of T24 and 5637 cells.

We employed Western blotting to detect the expression of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the two studied BC cell lines, which revealed that LBP can reduce the expression of Bcl-2, increase that of Bax, and activate caspase-3 and cleaved caspase-3 in both cell lines, thus confirming the efficacy of LBP in inhibiting proliferation and inducing apoptosis in the two BC cell lines. Bcl-2 is a protein in the Bcl-2 family that inhibits apoptosis. It is related to the survival, antiapoptotic activity and chemotherapy resistance of tumor cells and is also an important downstream effector in the PI3K/Akt pathway. It has been demonstrated that LBP inhibits the proliferation and migration of prostate cancer by regulating Bcl-2 and Bax (23). It also synergistically regulates cleaved caspase-3 to inhibit Ishikawa endometrial cancer cells (24). Anti-esophageal squamous cancer activity occurs through caspase-dependent apoptosis and enhanced Bax/Bcl-2 (10). This ability to adjust Bcl-2 and Bax expression also allows LBP to play a role in inhibiting the proliferation of human gastric cancer cells (25). The antiapoptotic PI3K/Akt pathway can cause an abnormal increase in cancer cells and accelerate the formation of tumors (26-30), and its PI3Kδ-specific inhibitor idelalisib (Zydelig^®) received good feedback in phase III clinical trials and is already available in the United States as an oral agent for the treatment of B-cell hematological cancer (31-33). The proapoptotic protein Bax is in the Bcl-2 family, and Bcl-2 can combine to form heterodimers that induce the release of cytochrome C (Cyt-c) and inhibit the antiapoptotic function of Bcl-2, therefore promoting cell apoptosis. The regulation of the proliferation of different tumor cells by LBP through the PI3K/Akt pathway has been well studied. For example, Pan et al. found that LBP can regulate the proliferation of esophageal squamous cell carcinoma through this pathway (34). Furthermore, as the most critical apoptosis executor downstream of the caspase cascade reaction, Bcl-2 activation largely depends on the release of Cyt-c (35,36). By triggering Cyt-c release through the mitochondrial pathway, Bcl-2 and Bax can influence activation of the downstream caspase-3 protease and then regulate the caspase cascade reaction to affect the survival or apoptosis of cells.

We detected protein expression levels in the two types of bladder cells before and after LBP treatment and found that, while LBP can reduce the expression of PI3K, Akt and the phosphorylated protein p-Akt in T24 cells, it increases the expression of PI3K and p-Akt and does not significantly reduce the expression of Akt in 5637 cells. Considering the important role of p-Akt in the functions of AKT (37), more specific studies are needed to discover more facts about the pathway. The occurrence and development of BC is associated with the overactivation of protooncogenes and the loss of cancer suppressor genes, as well as with changes in multiple signaling pathways in tumor cells (38,39). As a main signaling pathway for protein synthesis, the PI3K/Akt pathway is involved in the growth, proliferation, division and migration of cells (40). Dysregulation of the PI3K/Akt pathway can cause various diseases, such as cancer, cardiovascular diseases, metabolic disorders, and immune dysfunction. In MIBC, the PI3K/Akt pathway is overactive, and in 72% of BC cases, changes in the PI3K/AKT pathway can be observed (41,42). PI3K is an esterase, and after being activated by cytokines and growth factors, it can phosphorylate phosphatidylinositol-3,4-bisphosphate and PIP2 in the intracellular membrane to form phosphatidylinositol-3,4,5-bisphosphate and PIP3 and act as the second messenger to activate Akt. Then, PI3K works with the downstream factor Akt to inhibit the apoptosis of tumor cells and promote their proliferation and migration (43). The function of the PI3K/Akt signaling pathway makes it an attractive target for BC treatment (40).

Apart from the aforementioned results, we wanted to understand whether LBP can effectively inhibit cell proliferation in vivo, so we implanted T24 and 5637 tumor xenografts in nude mice for further evaluation of the antitumor activity of LBP in vivo. The results showed that LBP treatment can significantly reduce the size of bladder tumor tissues. The immunohistochemical method verified the effectiveness of LBP in raising the expression level of the proapoptotic proteins Bax and cleaved caspase-3 and lowering the expression level of the antiapoptotic protein Bcl-2, indicating that LBP-induced cell apoptosis contributes to inhibiting the growth of bladder tumors.

Conclusions

The experiments in this study confirmed that LBP is effective in inhibiting the proliferation and suppressing the migration of T24 and 5637 BC cells both in vivo and in vitro and is potentially able to promote the apoptosis of BC cells through the PI3K/Akt pathway. However, the underlying molecular mechanism is still unknown. Further research must be conducted to evaluate the impacts of LBP on BC cells. In summary, this study lays a foundation for the application of LBP to treat BC.

Acknowledgments

We thank Dr. Giorgio Ivan Russo (University of Catania, Catania, Italy), and Dr. Hiroshi Fukushima (Tokyo Medical and Dental University, Tokyo, Japan) for the critical comments and valuable advice on this study.

Funding: This work was supported by the special fund for clinical research of Wu Jieping Medical Foundation (No. 320.6750.19089-41) and Jilin Provincial Technological Development Program (No. 20160101032JC).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-23-376/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-376/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-376/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-23-376/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The in vivo experiment was performed according to the Guide for the Care and Use of Laboratory Animals of Jilin University and was approved by the ethics committee of Jilin University School of Pharmaceutical Science (No. 20190055).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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