Humic Acid Attenuates DSS-Induced Colitis, Increases MUC-2 Expression, and Restores Colonic Histoarchitectural Damage in Adult Male Wistar Rats

doi:10.21203/rs.3.rs-4660724/v1

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Humic Acid Attenuates DSS-Induced Colitis, Increases MUC-2 Expression, and Restores Colonic Histoarchitectural Damage in Adult Male Wistar Rats

https://doi.org/10.21203/rs.3.rs-4660724/v1

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This study investigates the role of humic acid (HA) in attenuating Dextran sulfate sodium (DSS)-induced UC in male Wistar rats. Twenty male Wistar rats were randomly assigned to groups (n = 5). Group 1 served as controls; Group 2 received 5% DSS alone; Group 3 received 5% DSS followed by humic acid (30 mg/kg); and Group 4 received 5% DSS followed by sulfasalazine (200 mg/kg). DSS was administered orally to induce colitis in Groups 2, 3, and 4. Colitis was induced for five days and drug treatment done for another 5 days Disease activity index was assessed on days 1, 3, 5, and 10. Animals were euthanized by cervical dislocation, colon specimens harvested for macroscopic assessment, histological and biochemical assays. Data were analyzed using one way ANOVA (p < 0.05). Results show humic acid treatment significantly attenuated DSS-induced colitis by reducing inflammation markers (TNF-α, interleukin-6, interleukin-1β, and arginase), lipid peroxidation (MPO) and restoring colonic histological integrity. Significant improvements were observed in colonic tissue nitrite levels and Muc-2 expression. Conclusively, HA shows promise as an alternative therapeutic for UC, offering antioxidant, anti-inflammatory, and mucosal barrier-protective effects.

Biological sciences/Biochemistry

Biological sciences/Physiology

Health sciences/Diseases

Health sciences/Gastroenterology

Inflammatory bowel diseases

humic acid

arginase

cytokines

Muc-2

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that damages the lining of the colon and the rectum causing inflammation, ulcers and bleeding in the gastrointestinal tract [1]. UC symptoms can range in severity and vary depending on the individual. Common symptoms are often diarrhea, abdominal spasms and pain, weight loss, exhaustion and anemia [2].

It is well established that UC is not an uncommon disease, as there are about a hundred thousand cases each year. The frequency and incidence of inflammatory bowel disease (IBD), a disease that affects people all over the world, are rising sharply [3]. The global incidence projected to reach 5 million cases by 2023 and continuing to rise [4]. Although, the manifestation of IBD is higher in developed countries compared to African countries, there is increasing reported cases in Africa [5]. This trend has been attributed to adoption of western diets by Africans. Western diets which are often processed foods are known to irritate the lining of the colon leading to inflammation and activation of cascade of immune responses in UC patients [6]. The disease's etiology is unknown, but a number of factors, including genetics, the environment, microbiology, diet and immunity, have been linked to its pathogenesis such that overreaction to the immune system may result in the release of mediators like cytokines and neutrophil infiltration, which may cause damage to the colon [1, 7].

Dietary changes and use of medications such as aminosalicylates, steroids, immunosuppressants, antibodies and anti-tumor necrosis factor (TNF)-alpha have been helpful in the treatment of IBD [8, 9] but they are not totally effective treatments. In addition, treatment response varies in individual [10]. Till today, plants and other organic products continue to serve as therapeutic agents to cure human diseases [11], especially in the rural regions of sub-Saharan countries where there are limitations to accessing medical facilities and abject poverty, which makes the dwellers unable to afford orthodox medicine. Hence, there is a need for a conventional way to treat illnesses such as ulcerative colitis with readily available nutraceuticals and novel bioactive compounds, such as humic acid.

Humic acid is a chemical produce by decaying plant and occur naturally in water, peat, soil and brown coal.Humic substances have been reported to possess anti-inflammatory as well as pro-inflammatory properties [12, 14]. In the recent time, the ethno-pharmacological potency of humic acid has been reported, especially in other lower animals [13]. With this growing body of knowledge, little is known about its effects on ulcerative colitis, and its effects on colitis have not been scientifically established. This study aimed at investigating the ameliorative effect of humic acid on immune response, inflammation and oxidative stress associated with dextran sulphate induced ulcerative colitis in wistar rats.

Drugs and Chemical

Ketamine hydrochloride (Ciron Drugs & Pharmaceuticals, India), Triochloroacetic acid (TCA, Sigma, Germany), Thiobarbituric acid (TBA, Sigma, Germany), 5, 50 dithio-bis-2-nitrobenzoic acid (DTNB, Sigma, Germany), Hexadecyltrimethyl ammonium bromide (HTAB, Sigma, Germany) and Adrenaline (Sigma, Germany). Tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) were carried out by ELISA kits from Biolegend, USA.

Extraction and purification of humic acid.

Humic acid was extracted by weighing 1kg of each of the soil samples into conical flasks, and 0.1 m NaOH was added to each of the soil samples, shaken for about 30 minutes with the help of a shaker. After 24 hours, the aqueous solution was centrifuged at 4000rpm for 15 minutes, to remove suspended particles and filtered. Then, the soil residue was washed with 500ml of 0.1 m NaOH, shaken for 15 minutes, and centrifuged at 4000rpm. The process was repeated till supernatant became clear. The supernatant was acidified to pH 2 with 0.1M HCl to induce the precipitation of humic acids and allowed to stand overnight. The supernatant, which contain fulvic acid, was separated from the precipitate by centrifugation. The humic acid was then redissolved in 0.1 m NaOH and centrifuged to completely remove clay impurities. The mixture was then acidified with 0.1M HCl to precipitate humic acid which was then washed with distilled water to remove other impurities that might be present. The precipitated humic acids were finally dialyzed in dialysis bag to remove the salts introduced during the extraction procedure against distilled water until chloride ion was no longer detected with AgNO₃ solution.The dialysed Humic acids were dissolved in ethanol to separate the insoluble humic acid from Hymatomelanic acid (soluble) which was present in the mixture, and centrifuged to remove the insoluble humic acid. The purified humic acid was dried in a controlled oven at 40 degree Celsius and kept for use when needed.

EXPERIMENTAL ANIMALS

Twenty (20) adult male Wistar rats with average weight of 160 ± 10 g were purchased Mctemmy animal farm. Animals were acclimatized for a month, allowed access to food and water freely. Animals were kept in ventilated plastic cages cushioned with wood shavings and housed in the animal facilities of the University of Medical Sciences, Ondo, Ondo State, Nigeria. Proper hygiene was also observed in their housing in accordance with the public health service policy on human care and the use of laboratory animals, approved by the Institute of Laboratory Animal Resources, National Research Council (2011). All procedures involving the use of animals conformed with the Animal Research: Reporting of in Vivo Experiments (ARRIVE) guidelines (2010) and ethical standards of the University of Medical Sciences Animal Care and Use.

Experimental Design

Twenty (20) adult male Wistar rats were divided into 4 groups as shown in the table below:

Table 1

Animal grouping
GROUPS	NUMBER OF ANIMALS	TREATMENT
1	5	No treatment
2	5	Dextran sulfate sodium
3	5	DSS + 30mg/kg of methanol extract of Humic acid
4	5	DSS + 200mg/kg of Sulfasalazine

As shown in Table 1 above, the rats in group 1 serves as control and did not receive any treatment throughout the study. The rats in group 2, 3 and 4 were given 5% DSS orally for five (5) days according to their body weights. Thereafter, they received distilled water, 30mg/kg of humic acid and 200mg/kg of sulfasalazine for five for five (5) days respectively.

On day eleven (11), they were euthanized by cervical dislocation and colon samples were collected in a Plain Sample bottle and homogenized in phosphate buffer saline (PH 7.4) and stored in -4 degrees Celsius. Tissue samples of the colon for histological and immunohistochemistry study were collected and stored in 10% formalin.

Induction of colitis

Colitis induction done in accordance with slightly modified method [37], using 5% (weight per volume) dextran sodium sulfate obtained from Sigma-Aldrich (United States). DSS was added to the drinking water for 5 days. Each drinking solution was administered to the rats in a bottle containing an equal number of moles of the compounds under study. Sulfasalazine was purchased from an accredited pharmaceutical store in Ondo State, Nigeria. Both humic acid and sulfasalazine were solubilized in distilled water as vehicles for oral administration. Humic acid (30 mg/kg) was administered to the rats orally based on their total body weight. Sulfasalazine was administered at a dose of 200 mg/kg via oral gavage.

Disease activity index (DAI)

The Disease Activity Index (DAI) was assessed on day 1, 3, and 5 of DSS treatment, as well as on day 10 equivalent to post-drug treatment period. Parameters used for assessment included stool consistency, diarrhea, and bleeding scores, which were obtained from observations. Consistent with the methodology described by Cooper et al.,[41], the DAI was calculated using the formula: DAI = (body weight drop + stool consistency + rectal hemorrhage) / 3. The humane endpoint was defined as DAI = 3, while a diagnosis of ulcerative colitis (UC) was established when DAI was ≥ 1.5.

Biochemical assessments of inflammation

Excised colon sections of rats were homogenized in sodium phosphate buffer (pH 7.4, 0.1M) and centrifuged at 4^oC at a speed of 1 x 10⁴ rpm for 6 x10² secs to obtain the supernatants which were thereafter stored at -20^oC.

Determination of Nitrite levels in the Colon tissue homogenate

Nitrite in the colon tissue homogenate was measured as an indicator of nitric oxide (NO) production according the Griess method as described by Green et al., [38]. The concentration of nitrite was determined from sodium nitrite standard curve and expressed as µmoles /mg protein.

Determination of Myeloperoxidase (MPO) activity in colon tissue homogenate

Tissues were suspended in extraction buffer 0.5% hexadecyltrimethylammonium bromide 50 Mm potassium phosphate buffer (pH 6.0) and frozen at 20°C. The process of freeze-thaw and sonication for 10 seconds cycle was repeated three times. The suspension was finally centrifugated at 15,000 rpm at 4°C for 15 min. MPO activity was assayed by adding 20 µL of supernatant to 96-microtiter plate, then 180 µL of reaction buffer (containing 0.167 mg/mL O-dianisidine in 50 mM potassium phosphate buffer and 0.15 mM H₂O₂) was added. The change in absorbance at 450 nm was monitored over 5 min in a microplate reader (LT4500, UK). One unit of MPO was defined as that giving a change in absorbance of 0.001 per min and the specific activity expressed as unit of MPO per milligram of protein.

Enzyme linked immunosorbent assay (ELISA) for determination of IL-6 and TNF-α in the colon tissue.

Colon tissue levels of IL-6 and TNF- α were determined by the Biolegend ELISA kit, (USA) specific to the cytokines of interest, with sensitivity limit of 4 pg/mL. All the measurements were done at room temperature in accordance to Biolegend instructions using microplate reader with 450nm filter. The concentration of IL-6 and TNF-α from the serum were extrapolated from the standard curves of IL-6 and TNF-α standards included in the assay kits and expressed as pg /mL.

The assay was carried out according to the protocol ELISA kit manufacturer, Biolegend ®, U.S.A.

Histological assessment of the colon

Samples of the colon tissue were processed for colitis estimation by an investigator shaded to study design and analysis was described [39]. In brief, the tissues were fixed in formalin (10%). Water was removed in graded alcohol. Thereafter, it was cleared in xylene and fixed in paraffin wax. The tissues were later cut into sections (four micrometer thick) by a microtome, embedded on the slides and stained with hematoxylin and eosin (H&E). The resultant slides were examined underneath a light microscope (Olympus, Japan) and photomicrographs were taken with DM750 camera (Leica, Germany) at 100 magnifications.

Immunohistochemistry and image quantification

Sections of 5 µm thickness obtained from routine paraffin were deparaffinized, and subjected to antigen retrieval by heating in a citrate-based antigen unmasking solution, pH 6.0 (Vector Labs, CA, USA) for 30 mins in a steamer and allowed to cool on the bench at room temperature for 30 mins. Endogenous peroxidase blocking was performed in 0.3% hydrogen peroxide in Phosphate Buffered Saline (PBS, pH 7.4) for 10 mins. Sections were then incubated at room temperature for 2 hours in primary rabbit antibodies diluted in a universal antibody diluent and blocking reagent, UltraCruz® Blocking Reagent (Santa Cruz, USA). Primary antibody used is MUC2 polyclonal antibody (Elabscience, USA; #E-AB-70212) at 1:500. Sections were washed in PBS and incubated in ImmPRESS™ HRP Anti-Rabbit IgG (Peroxidase) Polymer Reagent, made in horse (Vector Labs, USA). Colour was developed with DAB Peroxidase (HRP) Substrate Kit (Vector Labs, USA), and sections were counter-stained in hematoxylin [40].

Statistical analysis

This study utilized GraphPad Prism version 9.0.5 (GraphPad Software, San Diego, USA) for the analysis of collected data. The data was presented as mean ± standard error of mean (SEM) with n = 5. An analysis of variance (one-way ANOVA) was employed to examine mean differences, and the Tukey post hoc test was used for multiple comparisons. A significance level of P < 0.05 was considered statistically significant.

Effect of humic acid on the Disease activity index of Dextran sulfate sodium-induced colitis in adult male wistar rats.

As shown in Table 2 below, the results of the Disease Activity Index (DAI) across the days in each group indicated the progressive effect of DSS on colitis induction and effect of humic acid as well. There was statistical significance between the Disease activity index of day 3, 5 and 10 post-colitis induction. However, treatment with humic acid and sulfasalazine significantly reduced the DAI by day 10.

Table 2

Disease activity index (DAI) in rat groups; ^astatistically different from control (p < 0.05) ; ^bstatistically different from DSS only across the groups for each day.
DAYS	CONTROL	DSS only	DSS + HA (30 mg/kg)	DSS + SULFASALAZINE (200 mg/kgmg/kg)
1 post colitis induction	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
3 post colitis induction	0.00 ± 0.00	0.7330 ± 0.1400^a	0.6328 ± 0.1332^a	0.796 ± 0.1231^a
5 post colitis induction	0.00 ± 0.00	2.266 ± 0.1496^a	2.200 ± 0.1334^a	2.194 ± 0.1334^a
10 (treatment)	0.00 ± 0.00	2.346 ± 0.0988	0.230 ± 0.00^b	0.5666 ± 0.1666^ab

Effect of Humic acid on the macroscopic assessment of Dextran sulfate sodium-induced colitis in adult male wistar rats.

As shown in Fig. 1 below, a colon sample of a rat in the control group did not show any sign of colitis (Plate A). Oral administration of Dextran sulfate sodium (DSS) evoked a colonic inflammation as shown in Plate B. In contrast to the Dextran sulfate sodium-induced colitis rats, rats treated with 30mg/kg of humic acid (Plate C) showed no observable signs of colonic inflammation. Similarly, rats treated with 200m/kg of Sulfasalazine exhibited mild colonic inflammation (Plate D).

Figure 1. Effect of the humic acid on the macroscopic assessment of the colon of Dextran sulfate sodium-induced ulcerative colitis rats.

Effect of Humic acid on the assays of tissue oxidative stress marker on Dextran Sulfate Sodium-induced ulcerative colitis

As shown in Fig. 2, our data indicated a significant (p < 0.05) increase in the colon tissue level of myeloperoxidase (MPO); markers of lipid peroxidation in the DSS-treated rat group compared to the control group. On the other hand, treatment with humic acid significantly(p < 0.05) decreased the myeloperoxidase level compared with the DSS-only group.

Effect of Humic acid on the assays of colon tissue nitrite level on Dextran Sulfate Sodium-induced ulcerative colitis

As shown in Fig. 3, our data indicated a significant (p < 0.05) decrease in the colon tissue level of nitrite in the DSS-treated rat group compared to the control group. However, treatment with humic acid significantly(p < 0.05) increased the nitrite level compared with the DSS-only group.

Effect of humic acid on levels of inflammatory markers in the colon tissues of Dextran sulfate sodium-induced colitis rats

As shown in Fig. 4a to d, our data indicated a significant (p < 0.05) increase in the colon levels of markers of inflammation (TNF-α, interleukin-6, interleukin − 1β and arginase) in DSS-treated rats compared to the control group. On the other hand, treatment with humic acid significantly (p < 0.05) decreased the levels of TNF-α, interleukin-6, interleukin − 1β and arginase compared with the DSS-only group.

Effect of humic acid on Muc2 Gene Expression in DSS induced Ulcerative Colitis in wistar rats

As shown in the Figs. 5 and 6, the immunochemistry of MUC-2 expression colon tissues revealed significant reduction (p < 0.05) in MUC-2 expression compared to control. Results also show obvious MUC2 expression in the colon of humic acid treated group as compared to DSS group.

IMMUNOHISTOCHEMISTRY AND IMAGE QUANTIFICATION

Photomicrographs were taken at x400 magnification. Image analysis was performed using Image J software (NIH, USA). Positive immunoexpression of MUC2 is noted by brown colour in tissues.

Histological assessment of humic acid effects on the colon of DSS-induced ulcerative colitis.

As shown in the micrograph, H&E histological staining at x 100 magnifcation reveals inflammatory infiltrate, crypt and lining epithelial damage in the colon of DSS treated rat while the cellular architecture of the colon of humic acid treated rat was fully restored.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by relapsing and remitting inflammation of the colonic mucosa [7]. The incidence of ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD) affecting the colon and rectum, is rising quickly in developing nations [1]. Current therapeutic strategies aim to reduce inflammation and maintain remission, but side effects and the need for long-term medication pose significant challenges [8]. Humic acid as a complex mixture of organic compounds that is gotten from decaying plants. Even though little is known about the pharmacological actions of humic acid but few emerging studies have shown that humic acid has immune boosting potential antioxidant, anti-inflammatory, and immunomodulatory properties [14, 15].

In this present study, after inducing ulcerative colitis with DSS, animals exhibited similar symptoms to IBD, including weight loss, mucosal ulceration, and inflammation [16]. We obtained the Disease Activity Index (DAI); a composite score that evaluates the severity of colitis based on weight loss, stool consistency, and rectal bleeding. As shown in Table 2, DSS administration induced significant colitis in rats, evidenced by a progressive increase in DAI scores. By day 3, 5, and 10 post-colitis induction, the DAI scores reflected the acute inflammatory response and tissue damage induced by DSS. This is consistent with the previous studies where DSS treatment was used to induce colitis [17, 18]. The results of the significant increase in the disease activity index of this study as presented brought great insights that the severity of colitis progresses with time and when untreated, could have impacted greatly on the gastrointestinal membrane architecture

The pathogenic mechanisms of inflammatory bowel diseases are multifaceted and associated with oxidative stress, unbalanced gut microbiota, and aberrant immune response [19]. Oxidative stress plays a critical role in its pathogenesis, contributing to mucosal damage and inflammation. Myeloperoxidase (MPO) is an enzyme produced by neutrophils during inflammation, serving as a marker for oxidative stress and neutrophil infiltration [20, 21]. The expression of Pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and arginase play pivotal roles in the inflammatory cascade of UC [9, 22]. As shown in Fig. 2, DSS treatment significantly increased MPO levels in colonic tissues compared to controls, reflecting heightened oxidative stress. As shown in Figs. 5a-d, DSS treatment significantly elevated the levels of these cytokines in colonic tissues, reflecting the inflammatory milieu characteristic of UC.

Interestingly, treatment with HA significantly reduced MPO levels, indicating its potent antioxidant properties. This reduction in MPO aligns with previous reports where HA mitigated oxidative stress in different inflammatory conditions [23, 24]. HA treatment significantly decreased the levels of TNF-α, IL-6, IL-1β, and arginase compared to the DSS-only group, highlighting its anti-inflammatory potential. These results are in agreement with previous studies that demonstrated HA's ability to downregulate pro-inflammatory cytokine production in various inflammatory models [25, 32]. Notably, treatment with HA significantly reduced the DAI by day 10, indicating their therapeutic potential in mitigating colonic inflammation. This reduction in DAI aligns with previous findings where Albiflorin and N-Acetyldopamine Dimer demonstrated anti-inflammatory effects in DSS- induced ulcerative colitis models [26, 27].

Nitrite levels in colonic tissues are indicative of nitric oxide (NO) production, a mediator of inflammation and tissue damage in UC [28]. As revealed in our study, DSS-induced colitis significantly decreased the nitrite levels compared to the control group, likely due to the consumption of NO during the inflammatory process. Treatment with HA significantly increased nitrite levels compared to the DSS-only group, suggesting a restoration of NO balance and potential modulation of inflammatory pathways. This finding is consistent with studies indicating that HA can modulate NO production and reduce inflammation [29].

Ulcerative colitis pathogenesis hinges on an imbalance where protective mucosal mechanisms fail against aggressive inflammatory responses in the colon. MUC-2 is a key component of the mucosal barrier, and its expression is critical for maintaining gut integrity [30]. In UC, MUC-2 expression is often reduced, compromising the mucosal barrier and exacerbating inflammation. Immunohistochemical analysis revealed a significant reduction in MUC-2 expression in the DSS-treated group compared to controls (Figs. 6 and 7). However, HA treatment restored MUC-2 expression, suggesting a protective effect on the mucosal barrier. This finding is supported by previous research indicating that HA can enhance mucosal barrier function and reduce intestinal permeability [31].

Macroscopic assessment in rat models of ulcerative colitis is relevant for evaluating gross pathological changes such as mucosal ulceration, bleeding, and tissue inflammation, providing crucial insights into disease severity and progression [33]. Our result of macroscopic assessment of the colon revealed distinct differences between the groups. The control group showed no signs of colitis, with healthy and intact colonic mucosa (Plate A). In contrast, DSS administration resulted in pronounced colonic inflammation, characterized by mucosal erythema, edema, and ulcerations (Plate B). Treatment with HA (30 mg/kg) markedly attenuated these macroscopic signs of inflammation, with only no obvious sign of colonic inflammation observed (Plate C). Similarly, sulfasalazine treatment (200 mg/kg) resulted in mild colonic inflammation (Plate D). These findings suggest that HA can significantly ameliorate the macroscopic damage induced by DSS, corroborating its protective effects as observed in other studies where natural products produced similar effects [34, 35].

The macroscopic restoration of the colonic histoarchitecture HA treated rats is a function of the anntioxidant and anti-inflmmatory effects HA which was further corroborated in the histological examination. Histological examination of colonic tissues gives insights into the cellular and structural changes induced by DSS and the protective effects of treatments [36]. Hematoxylin and eosin (H&E) staining not only revealed significant inflammatory infiltrate, but also crypt damage and epithelial disruption in the colon of DSS-treated group (micrograph). In contrast, HA-treated rats showed restored cellular architecture with reduced inflammation and crypt damage. These histopathological findings corroborate the macroscopic and biochemical data, underscoring the therapeutic potential of HA in UC.

In conclusion, the results of our study demonstrate that HA holds potential as an alternative therapeutic agent for UC, offering antioxidant, anti-inflammatory, and mucosal barrier-protective properties. Further studies are required to fully elucidate its mechanisms and optimize its therapeutic application in inflammatory bowel diseases.

Ethical Approval

Ethical approval was requested from the University of Medical Sciences Research and Ethics Committee on Animal use and care and granted with the approval reference UNIMED-AREC/Apv/2023/033.

Conflicting interests

There is no conflict of any kind to declare

Funding

No external funding was received.

Author Contribution

OBO and AGA conceived, designed, supervised the research and edited manuscript, OOE co-supervised the experiments, did literature search, analyzed data, and wrote the manuscript, AA, EK, AOT and AO conducted the experiment, AJK helped in the extraction and purification of the humic acid.

Acknowledgment

The authors express their gratitude to the technical personnel of the Animal House and the Department of Physiology, University of Medical Sciences, Ondo, Nigeria.

Data Availability

All data supporting the findings of this study are available within the paper.

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No competing interests reported.

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Humic Acid Attenuates DSS-Induced Colitis, Increases MUC-2 Expression, and Restores Colonic Histoarchitectural Damage in Adult Male Wistar Rats

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Abstract

Figures

Introduction

MATERIALS AND METHODS

Drugs and Chemical

EXPERIMENTAL ANIMALS

Experimental Design

Induction of colitis

Disease activity index (DAI)

Biochemical assessments of inflammation

Determination of Nitrite levels in the Colon tissue homogenate

Determination of Myeloperoxidase (MPO) activity in colon tissue homogenate

Histological assessment of the colon

Immunohistochemistry and image quantification

Statistical analysis

RESULTS

IMMUNOHISTOCHEMISTRY AND IMAGE QUANTIFICATION

Discussion

Conclusion

Declarations

Ethical Approval

Conflicting interests

Funding

Author Contribution

Acknowledgment

Data Availability

References

Additional Declarations

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