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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 3  |  Page : 150-158

Scopoletin and umbelliferone from Cortex Mori as protective agents in high glucose-induced mesangial cell as in vitro model of diabetic glomerulosclerosis


1 School of Medicine, Foshan University, Foshan, China
2 The R and D Center of Drug for Renal Diseases, Consun Pharmaceutical Group, Guangzhou, China
3 Foshan Newtopcome Pharmaceutical Technology Co., Ltd., Foshan, China
4 School of Medicine, Foshan University; Foshan Newtopcome Pharmaceutical Technology Co., Ltd., Foshan, China

Date of Submission31-Jan-2021
Date of Decision15-May-2021
Date of Acceptance19-May-2021
Date of Web Publication24-Jun-2021

Correspondence Address:
Dr. Zhaoguang Zheng
School of Medicine, Foshan University, No. 5 Road, Chancheng Area, Foshan; Foshan Newtopcome Pharmaceutical Technology Co., Ltd., No. 129 West Jihua Road, Chancheng Area, Foshan
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_9_21

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  Abstract 


Two known coumarins, scopoletin (SP) and umbelliferone (UB), were isolated from Cortex Mori (CM). Their structures were elucidated by various spectroscopic analyses. Then, their effects on rat glomerular mesangial cells (RGMCs, HBZY-1) proliferation, hypertrophy, extracellular matrix (ECM) proliferation, expression of fibronectin, transforming growth factor-beta (TGF-β), and connective tissue growth factor (CTGF) induced by high glucose were studied in vitro model of diabetic glomerulosclerosis. The results show that, CM, SP, and UB can inhibit the RGMCs proliferation to attenuate the ECM proliferation and cell hypertrophy, reduced the accumulation of ECM protein fibronectin, and lowered the expression of the key fibrosis factor TGF-β and CTGF to inhibit the kidney fibrosis and thereby improved diabetic glomerulosclerosis. The two coumarins show great potentialities on treating diabetic glomerulosclerosis, but the animal experiment and mechanism is strongly needed for further proof.

Keywords: Cortex Mori, diabetic kidney disease, glomerulosclerosis, proliferation, rat glomerular mesangial cells, scopoletin, umbelliferone


How to cite this article:
Liang Y, Zeng X, Guo J, Liu H, He B, Lai R, Zhu Q, Zheng Z. Scopoletin and umbelliferone from Cortex Mori as protective agents in high glucose-induced mesangial cell as in vitro model of diabetic glomerulosclerosis. Chin J Physiol 2021;64:150-8

How to cite this URL:
Liang Y, Zeng X, Guo J, Liu H, He B, Lai R, Zhu Q, Zheng Z. Scopoletin and umbelliferone from Cortex Mori as protective agents in high glucose-induced mesangial cell as in vitro model of diabetic glomerulosclerosis. Chin J Physiol [serial online] 2021 [cited 2021 Dec 9];64:150-8. Available from: https://www.cjphysiology.org/text.asp?2021/64/3/150/319286




  Introduction Top


According to the International Diabetes Federation, the prevalence of diabetes in the world is estimated to increase from 285 million persons to 439 million in 2030.[1] Diabetic kidney disease (DKD) develops in approximately 40% of patients who are diabetic and is the leading cause of chronic kidney disease worldwide.[2] All forms of diabetes are characterized by hyperglycemia. Hyperglycemia is the primary pathogenic factor for DKD.[3] Through multiple mechanisms, DKD can develop to end-stage kidney disease, but none is as important as the gradual, inexorable scarring of the renal glomerulus, known as glomerulosclerosis.[4] Glomerulosclerosis is DKD caused by accumulation of extracellular matrix (ECM) proteins in mesangial interstitial space, resulting in fibrosis manifested by either diffuse or nodular changes.[5] One of the most common matrix proteins detected is fibronectin.[4] Several studies also found that hyperglycemia induces reactive oxygen species (ROS) production in mesangial cells that upregulate transforming growth factor-beta (TGF-β) and connective tissue growth factor (CTGF) involved in ECM accumulation.[4],[6]

Cortex Mori (CM), the root bark of Morus alba L., is a famous traditional Chinese folk medicine. With the effect of facilitating the circulation of lung-Qi to relieve asthma and inducing diuresis to remove edema, CM has been regularly applied to anti-inflammatory and analgesic,[7],[8],[9] antitussive and antiasthmatic,[10] diuretic,[11] hypoglycemic,[12] hypolipidemic,[13],[14] blood pressure lowering,[15] peripheral neuropathy,[16],[17] antitumor,[18] antivirus,[19] and so on. In the recent years, although more and more effects and mechanism of CM have been reported for the treatment of diabetes mellitus and its complications such as peripheral neuropathy[16],[17] and DKD,[20] the chemical material basis remains unknown.

High glucose (HG, 20–30 mM) stimulates renal tubular epithelial cells and mesangial cells to produce massive ROS and induces the damage of renal tubulars and the change of ECM.[21] In the present study, we established the glomerulosclerosis model by culturing rat glomerular mesangial cells (RGMCs) with HG (25 mM) in vitro[22] and treated with Cortex Mori extraction (CME) and two coumarins from CM. The results show that CME, scopoletin (SP), and umbelliferone (UB) could significantly inhibit the RGMC proliferation, hypertrophy, and ECM proliferation, and meanwhile, reduced the expression of fibronectin, TGF-β1, and CTGF.


  Materials and Methods Top


Chemicals and reagents

The established RGMC line HBZY-1 was obtained from the Chinese Center for Type Culture Collection (Wuhan, China). The microtetrazoliumpowder (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]) was purchased from Amresco (Solon, OH, USA). HEPES, penicillin, and streptomycin were purchased from FlowLab (Sydney, Australia). Fetal calf serum was obtained from Sijiqing Serum Factory (Hangzhou, China). Tips, dishes, test tubes, etc., for cell culture were bought from Thermo Fisher Scientific (Waltham, MA, USA).

Plant material

CM were collected from Bozhou, Anhui Province, P. R. China, during November 2017 and identified by Prof. Quan Zhu. A voucher specimen (CM 20171121) was stored at the Laboratory of Pharmaceutical, School of Medicine, Foshan University.

Extraction and isolation

Ten-kilogram CM were ground into power, extracted by reflux with 100 L water for three times (1 h per time), combined the extract, concentrated to an appropriate volume under reduced pressure, then added to D101 macroporous resin, and sequentially eluted with water and 95% ethanol. The 95% ethanol eluent was collected, concentrated under reduced pressure to obtain 150 g of dry extract; then 130 g dry extract was applied to silica gel column and eluted by gradient elution of petroleum ether-ethyl acetate and chloroform-methanol, respectively. The fractions eluted with petroleum ether-ethyl acetate were purified repeatedly by silica gel and Sephadex LH-20 CC to obtain SP (100 mg, >98% by high-performance liquid chromatography [HPLC]) and UB (80 mg, >98% by HPLC).

Biological experiment material

Preparation of Cortex Mori extraction

Ten-gram CM were ground into power, extracted by reflux with 100 mL water for three times (1 h per time), combined the extract, and concentrated to an appropriate volume under reduced pressure.

Cell culture

The RGMCs HBZY-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL), supplemented with 10% fetal calf serum, 15 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 mM glucose at 37°C in an atmosphere containing 5% CO2/95% air. Cultures were passaged twice weekly at a 1:4 split with the abovementioned medium. Then, cells were cultured in 96-well plates at densities of 1 × 104 cells/well in DMEM (5 mM glucose) supplemented with 5% fetal calf serum until subconfluence was attained. Cell growth was arrested by reducing the fetal calf serum content of the medium to 0.5%. After 24 h, either the cells were left unstimulated (5 mM glucose), or growth was initiated by DMEM containing 25 mM glucose with 0.5% fetal bovine serum (FCS) in the presence or absence of CME (final concentration 0.1 g/L), SP, UB (final concentration 0.01 μM, 0.1 μM, and 1 μM), or 20 mM alpha-tocopherol (purity: 96.7% by HPLC; Sigma-Aldrich) as a positive control. The plates were then incubated for 48 h.

Cell proliferation assay

The inhibition of cell proliferation induced by HG was assessed by a standard MTT-based colorimetric assay.[23] Briefly, after the plates were incubated for 48 h, proliferation was examined by MTT reduction assay. Experimental conditions were tested in sextuplicate (six wells of the 96-well plate per experimental condition). All of the experiments were performed in triplicate. The cell proliferation rate (%) was calculated as follows: cell proliferation rate (%) = (Optical density (OD) value of each group) × 100/(OD value of control group).

Hematoxylin and eosin/periodic acid–Schiff staining

After the plates were incubated for 48 h, routine hematoxylin and eosin and periodic acid–Schiff (PAS) stains were used to observe the cell hypertrophy and ECM proliferation by an inverted microscope (Chongqing Optical Instrument Factory).

Quantification of fibronectin level

The quantitative determination of fibronectin level in the cell-free supernatant was performed using Rat FN (Fibronectin) ELISA Kit (ElabSci E-EL-R0578) based on manufactured protocol. Briefly, 100 μl of standard, blank, and sample solution was added into each well and then sealed and incubated for 90 min at 37°C. The cell-free supernatant, after treated with CME, SP, and UB, was served as the sample. The glucose-induced mesangial cell-free supernatant without extract and compounds was used as positive control (HG group). The normal cell or untreated cell was used as negative control. Subsequently, the liquid of each well was discarded and 100 μl of biotinylated detection antibody was added and incubated for an hour at 37°C. The liquid was discarded again and the plate was washed three times using 200 μl wash buffer. Horseradish peroxidase conjugate (100 μl) was added and incubated for 30 min at 37°C. The liquid was discarded again and the plate was washed five times using 200 μl wash buffer. Substrate reagent (90 μl) was added and incubated for 15 min at 37°C. Stop solution (50 μl) was added and the absorbance was measured at 450 nm using Multiskan GO plate reader (Thermo Scientific, USA).[22]

Immunocytochemistry assay of connective tissue growth factor and transforming growth factor-beta 1

The streptavidin–biotin enzyme complex (SABC) method was applied to evaluate the effect of CME, SP, and UB on the CTGF and TGF-β1 expressions in HBZY-1 cells. After being incubated with drugs as indicated above, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 90 min at room temperature. Then, cells were blocked with 10% primary antibody-origin serum for 20 min and incubated with CTGF (1:500, Canta Cruz) and TGF-β1 (1:400) antibodies for 120 min at 37°C. The cells were sequentially incubated with biotin-conjugated secondary antibody (Jingmei Bioengineering Co., Ltd.) for 30 min and SABC (Boster Biological Technology Co. Ltd.) for 20 min. Finally, 0.3 mg/mL 3,3'-diaminobenzidine (Gene Tech Biotechnology Company Limited) was added to the coverslips for colorization, and the slip was counterstained with hematoxylin and photographed under microscope with Image-Pro Plus picture analysis software. The primary antibody was replaced with PBS as negative control.

Statistical analysis

All values were expressed as mean ± standard deviation, and the analysis software was GraphPad Prism software (Version 7.0, GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance followed by Tukey's post hoc test was used to analyze the statistical significance among multiple groups. P < 0.05 was considered to indicate statistical significance.


  Results Top


Isolation and identification of scopoletin and umbelliferone

Two known coumarins SP and UB were isolated from CM and identified by various spectroscopic analyses. Their structures [Figure 1] were similar with a more 6-OMe substituent in SP.
Figure 1: Structures of scopoletin and umbelliferone.

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Scopoletin

Pale yellow powder, ESI-MS m/z: 193 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ:10.33 (1H, s, OH), 7.91 (1H, d, J = 9.5 Hz, H-4), 7.26 (1H, s, H-5), 6.78 (1H, s, H-8), 6.26 (1H, d, J = 9.5 Hz, H-3), 3.90 (3H, s, CH3); 13C NMR (CDCl3, 100 MHz) δ:161.4 (C-2), 150.3 (C-7), 149.7 (C-9), 144.0 (C-6), 143.3 (C-4), 113.4 (C-3), 111.5 (C-10), 107.5 (C-5), 103.2 (C-8), 56.4 (-OCH3).

Umbelliferone

Pale yellow powder, ESI-MS m/z: 163 [M + H]+; mp. 226 ~ 228; 1H NMR (CD3OD, 400 MHz) δ: 7.82 (1H, d, J = 9.2 Hz, H-4), 7.43 (1H, d, J = 8.5 Hz, H-5), 6.78 (1H, d, J = 8.5, 2.3 Hz, H-6), 6.69 (1H, d, J = 2.3 Hz, H-8), 6.16 (1H, d, J = 9.5 Hz, H-3); 13C NMR (CD3OD, 100 MHz) δ: 163.7(C-2), 163.1 (C-7), 157.3 (C-9), 146.0 (C-4), 130.7 (C-5), 114.5 (C-3), 113.2 (C-10), 112.4 (C-6), 103.4 (C-8).

Effects of umbelliferone and scopoletin on rat glomerular mesangial cell proliferation, hypertrophy, and extracellular matrix proliferation

Compared with the normal control group, the RGMCs induced by HG (25 mM) proliferated markedly [Figure 2]. When treated with tocopherol, CME, SP, or UB, the cell proliferation was inhibited. Except the low concentration groups (0.01 μM) of SP and UB, all the other groups inhibited the proliferation significantly (P < 0.05). Both SP and UB were in a clear dose-dependent manner. In order to observe the cellular morphology such as hypertrophy and ECM proliferation, after the plates were incubated with HG for 48 h, routine HE and PAS stains were applied. When the RGMCs were stimulated by high glucose, the cell hypertrophy and extracellular matrix proliferation were observed. Treatment with CME, SP, or UB could improve the morphology [Figure 3] and [Figure 4], which was consistent with the effect on cell proliferation.
Figure 2: Effects of umbelliferone and scopoletin on rat glomerular mesangial cells proliferation induced by high glucose. ##P < 0.01, versus control group; *P < 0.05, **P < 0.01 versus high glucose group.

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Figure 3: Effects of umbelliferone and scopoletin on hypertrophy of rat glomerular mesangial cells induced by high glucose (H and E, ×200). (a) control; (b) high glucose (25 mmol/L); (c) Cortex Mori extraction (0.1 g/L); (d) umbelliferone (1 μmol/L); (e) umbelliferone (0.1 μmol/L); (f) umbelliferone (0.01 μmol/L); (g) scopoletin (1 μmol/L); (h) scopoletin (0.1 μmol/L); (i) scopoletin (0.01 μmol/L).

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Figure 4: Effects of umbelliferone and scopoletin on extracellular matrix proliferation of rat glomerular mesangial cells induced by high glucose (PAS, ×200). (a) control; (b) high glucose (25 mmol/L); (c) Cortex Mori extraction (0.1 g/L); (d) umbelliferone (1 μmol/L); (e) umbelliferone (0.1 μmol/L); (f) umbelliferone (0.01 μmol/L); (g) scopoletin (1 μmol/L); (h) scopoletin (0.1 μmol/L); (i) scopoletin (0.01 μmol/L).

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Effects on fibronectin, connective tissue growth factor, and transforming growth factor-beta expression

[Figure 5] shows that HG can stimulate overexpression of ECM protein in the RGMCs (P < 0.01 vs. control group) and CME, SP, or UB blocked it significantly (P < 0.01), and both SP and UB were in a clear dose-dependent manner.

In CTGF and TGF-β expression assay [Figure 6] and [Figure 7], HG induced the high expression of CTGF and TGF-β (P < 0.01). Except the 0.01 μM UB group, all the other groups of CME, SP, or UB reduced the expression significantly (TGF-β expression of CME and 0.1 μM UB groups, P < 0.05; other groups, P < 0.01), and both SP and UB were in a clear dose-dependent manner.
Figure 5: Fibronectin level of high glucose-induced HBZY-1 cells treated with Cortex Mori extraction, SP and UB. ##P < 0.01, versus control group; **P < 0.01 versus high glucose group.

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Figure 6: Effects of umbelliferone and scopoletin on connective tissue growth factor expression of rat glomerular mesangial cells induced by high glucose (connective tissue growth factor, ×200). (A) Immunocytochemistry staining of connective tissue growth factor expression: (a) control; (b) bull serum albumin (BSA); (c) high glucose (25 mmol/L); (d) Cortex Mori extraction (0.1 g/L); (e) umbelliferone (1 μmol/L); (f) umbelliferone (0.1 μmol/L); (g) umbelliferone (0.01 μmol/L); (h) scopoletin (1 μmol/L); (i) scopoletin (0.1 μmol/L); (j) scopoletin (0.01 μmol/L). (B) Connective tissue growth factor expression score. ##P < 0.01, versus control group; **P < 0.01 versus high glucose group.

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Figure 7: Effects of umbelliferone and scopoletin on transforming growth factor-beta expression of rat glomerular mesangial cells induced by high glucose (transforming growth factor-beta, ×200). (A) Immunocytochemistry staining of transforming growth factor-beta expression: (a) control; (b) BSA; (c) high glucose (25 mmol/L); (d) Cortex Mori extraction (0.1 g/L); (e) umbelliferone (1 μmol/L); (f) umbelliferone (0.1 μmol/L); (g) umbelliferone (0.01 μmol/L); (h) scopoletin (1 μmol/L); (i) scopoletin (0.1 μmol/L); (j) scopoletin (0.01 μmol/L). (B) Transforming growth factor-beta expression score. ##P < 0.01, versus control group; *P < 0.05, **P < 0.01 versus high glucose group.

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As a whole, the effect of SP is better than UB. The more 6-OMe substituent in SP helps to improve the effects.


  Discussion Top


M. alba L. used to raise silkworms is full of treasure. Folium Mori, Ramulus Mori, Fructus Mori, and CM, all from M. alba L., are famous Chinese folk medicine. Total alkaloids from Ramulus Mori have been approved to new drug for treating diabetes by the National Medical Products Administration of China in 2020. It has been reported that the extraction of CM prevents DKD through inhibition of inflammation and fibrosis in a rat model,[20] but the chemical material basis remains unknown. The major chemical constituents of CM include Diels–Alder adducts, terpenoids, flavonoids, coumarins, alkaloids, sterols, polysaccharides, stilbenoids, and essential oils. Total flavonoids, total alkaloids, and total polysaccharides of CM have been reported to regulate the blood sugar level, and total flavonoid is particularly significant.[24] The component of coumarins is less concerned at present. Coumarins, mainly including 5,7-dihydroxycoumarin, SP, and UB, are traditionally regarded as constituents of antitussive and antiasthmatic of CM.[24],[25] SP, about 4 mg/g in CM,[26] possesses a variety of pharmacological activities, including antitumor,[27] antihyperuricemic,[28] acaricide,[29] and so on. It has been reported that SP is able to counteract the effect of chronic and acute administration of angiotensin II, on hypertension, but also the inflammatory and oxidative damage in the kidney.[30] UB, another important coumarin, has been reported to ameliorate renal function in DKD rats through regulating inflammation.[31],[32] Moreover, UB can significantly reduce the diabetes-induced renal damage and improves the pathological conditions related to the DKD by downregulation of the renal tissue and circulating TGF-β,[33] which is consistent to our current studies.

DKD is one of the most important long-term complications of diabetes and the main cause of end-stage renal disease, which often has a progress of 10–20 years.[34] The pathological characteristics of DN included the proliferation of mesangial cells and accumulation of ECM.[35] Ke et al.[36] demonstrated that in glomerular mesangial cells, HG induced an increased synthesis of ECM proteins, including laminin, fibronectin, and collagen IV. Moreover, these pathological changes would promote progressive diabetic glomerulosclerosis and renal function damage.[37] Renal fibrosis, the final common pathway of numerous progressive kidney diseases, is characterized by glomerulosclerosis and tubulointerstitial fibrosis with an increase in mesangial cell proliferation, ECM protein production, a decrease in matrix degradation, dysregulation of cell-matrix interaction, inflammatory cell infiltration, and transformation of resident cells.[38] Various cytokines and growth factors are reportedly involved and associated with fibrogenic and inflammatory processes. Of these, TGF-β has been shown to play a central role in the development of renal fibrosis.[39] TGF-β, a multifunctional dimeric peptide, regulates biological processes such as cell proliferation, differentiation, and immunological reaction. One of the most important biological actions of TGF-β is the regulation of ECM accumulation.[38] TGF-β upregulates the synthesis of individual matrix components, including collagens, proteoglycans, and glycoproteins. CTGF, the downstream mediator of TGF-β, is a member of the CCN matricellular protein family, consisting of four domains, that regulates the signaling of other growth factors and promotes kidney fibrosis.[39]

Evidences have strongly suggested that the inhibition of TGF-β action should be viable herapeutic strategies to prevent the progression of renal fibrosis. Many strategies targeting TGF-β, including inhibition of production, activation, binding to the receptor, and intracellular signaling, have been developed.[38] CTGF, the downstream mediator of TGF-β, plays an important role in the renal fibrosis. CTGF expression in fibrosis is also reported to occur in the kidney area. Exposure of mesangial cell to recombinant human CTGF significantly increased production of fibronectin and collagen type I. Induction of CTGF in rat mesangial cells due to HG levels is mediated by TGF-β. The study of human kidney biopsy samples from various kidney diseases has revealed that CTGF expression level is increased in glomerulosclerosis and tubulointerstitial fibrosis.[39] An interventional study of an animal model is first reported by Yokoi et al.[39] Treatment of CTGF antisense oligonucleotide markedly attenuates the induction of fibronectin and collagen expressions in the rat unilateral ureteral obstruction model.[39] Another study also showed the efficacy of CTGF inhibition by CTGF antisense oligonucleotide in subtotal nephrectomy of TGF-β transgenic mice.[40]

In our present study, CME, SP, and UB can inhibit the RGMC proliferation. Except the low concentration groups (0.01 μM) of SP and UB, all the other groups inhibited the proliferation significantly. Both SP and UB were in a clear dose-dependent manner. Moreover, they can inhibit the hypertrophy and ECM proliferation. In the accumulation of ECM protein, CME, SP, and UB reduced the production of fibronectin significantly (P < 0.01) and furthermore lowered the expression of key fibrosis factor TGF-β and CTGF. Hence, the two coumarins show great potentialities on treating diabetic glomerulosclerosis.


  Conclusion Top


The present study indicates that CM, SP, and UB can inhibit the RGMCs proliferation to attenuate the ECM proliferation and cell hypertrophy, reduced the accumulation of ECM protein fibronectin, and lowered the expression of the key fibrosis factor TGF-β and CTGF to inhibit the kidney fibrosis and thereby, improved diabetic glomerulosclerosis [Figure 8]. The two coumarins show great potentialities on treating diabetic glomerulosclerosis, but the animal experiment and mechanism is strongly needed for further proof.
Figure 8: Graphic summary of this study. GCMs: Glomerular mesangial cells, ECM: Extracellular matrix, TGF-β1: Transforming growth factor-beta 1, CTGF: Connective tissue growth factor, FN: Fibronectin, CME: Cortex Mori extraction, SP: Scopoletin, UB: Umbelliferone.

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Financial support and sponsorship

This work was financially supported by the NSFC (81901881, 81872832), the Science and Technology Key Project of COVID-19 in Foshan city (No. 2020001000206), Regional Joint Fund-Key Project of Guangdong Basic and Applied Basic Research fund (2020B1515120033), and the Scientific Research Program of High-Level Talents of Foshan University (No. CGZ07001).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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