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ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 1  |  Page : 35-40

Antiproliferative potential of methanolic extracts of Ageratum conyzoides linnaeus via downregulation of ki-67 and upregulation of p53 protein expression in hepatic tissue of rats


1 Department of Anatomy, College of Health Sciences, Igbinedion University, Benin City, Edo State, Nigeria
2 Department of Anatomy, College of Medicine, University of Benin, Benin City, Edo State, Nigeria

Date of Web Publication13-Sep-2019

Correspondence Address:
Dr. Dayo Rotimi Omotoso
Department of Anatomy, College of Health Sciences, Igbinedion University, Okada, Edo State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_21_19

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  Abstract 


Background: Hepatic tissue is susceptible to toxicity induced by hepatotoxins, leading to lesions, necrosis, fibrosis, tumor, or carcinoma. However, it is self-regenerating which can actively proliferate to recover lost segments. During tissue proliferation, molecular markers such as Ki-67 and p53 proteins usually play opposing roles. In this study, we aimed at assessing the antiproliferative potential of methanolic extracts of Ageratum conyzoides Linn using the expression of these markers of proliferation in hepatic tissues of adult male Wistar rats. Materials and Methods: Twenty animals used for this study were divided equally into four groups (1–4) as follows: Group 1 represented control, whereas Groups 2–4 were, respectively, treated with extracts at dosage of 100, 300, and 500 mg/kg (body weight) orally for 28 days. Afterward, the animals were sacrificed; their hepatic tissues were harvested, were processed into tissue sections, were histologically stained using hematoxylin and eosin technique, and were immunostained for Ki-67 and p53 proteins using horseradish peroxidase-3, 3-Diaminobenzidine technique (with monoclonal anti-Ki67 and anti-p53 antibodies). Stained sections were examined and quantified using Image-J software. Data obtained were statistically analyzed using IBM-SPSS (version 20) and compared using t- test. Results: The tissue histology showed densely packed hepatocytes in treated groups. The immunostaining revealed statistically significant (P < 0.05) upregulation of the Ki-67 expression only among the treated Group 4 animals, whereas the p53 protein expression was statistically significantly (P < 0.05) upregulated in all the treated groups. Conclusions: The findings of this study implied that only higher dose of extracts could trigger increase in the rate of hepatic cell proliferation even the inhibitory signal of proliferation becomes activated. Hence, the exposure of methanolic leaf extracts of A. conyzoides L. may cause dose-dependent antiproliferative activity.

Keywords: Ageratum conyzoides, antiproliferative, hepatic tissue, Ki-67 protein, p53 protein, rats


How to cite this article:
Omotoso DR, Eze GI. Antiproliferative potential of methanolic extracts of Ageratum conyzoides linnaeus via downregulation of ki-67 and upregulation of p53 protein expression in hepatic tissue of rats. Niger J Exp Clin Biosci 2019;7:35-40

How to cite this URL:
Omotoso DR, Eze GI. Antiproliferative potential of methanolic extracts of Ageratum conyzoides linnaeus via downregulation of ki-67 and upregulation of p53 protein expression in hepatic tissue of rats. Niger J Exp Clin Biosci [serial online] 2019 [cited 2020 Jan 20];7:35-40. Available from: http://www.njecbonline.org/text.asp?2019/7/1/35/266838




  Introduction Top


The basic structural component of the hepatic tissue, the hepatocytes, can be described as the most versatile cell due to its ability to synthesize, accumulate, and breakdown different substances. These activities are a function of its intracellular organelles which in turn form the basis for the strong eosinophilic and prominent basophilic granulity of the cell.[1],[2] As a major organ of metabolism, hepatic tissue is susceptible to toxicity induced by hepatotoxins which may be naturally occurring organic substances or ingested synthetic compounds with varying toxic effects such as lesions, necrosis, fibrosis, tumor, or carcinoma.[3] Often, hepatic injury results when reactive metabolites interact with cellular macromolecules, leading to lipid peroxidation, oxidative stress and DNA damage or disruption of ion transport and storage leading to mitochondrial dysfunction, and ultimately production of oxidants that cause cellular injury.[4] However, the hepatic tissue is a self-regenerating tissue which can actively proliferate to recover lost segments following tissue injury or necrosis or after surgical resection or partial hepatectomy.[5],[6] Due to the prominence of hepatocytes within hepatic tissue parenchyma, rapid proliferation of hepatocytes has been described to mainly account for hepatic tissue regeneration.[7],[8] During hepatic tissue regeneration, most hepatocytes are re-routed into the synthetic(S) phase of cell cycle prior to mitotic (M) phase. Furthermore, there is activation and proliferation of facultative stem cell population containing oval cells which rapidly proliferate and invade hepatic lobules to differentiate into hepatocytes and biliary epithelium.[9],[10],[11] Among the key markers of tissue proliferation are the cell proliferation (Ki-67) and tumor suppressor (p53) proteins. The Ki-67 is a nuclear protein encoded by Ki-67 gene, which is widely regarded as the marker of cell cycle and proliferation. The expression of the wild-type Ki-67 usually occurs in all phases of cell cycle except the resting (G0) phase in quiescent cells or during the process of DNA damage repair, and it is predominant in the nucleus where it plays a key role in the regulation of cell division.[12],[13],[14] Similarly, the p53 protein is a nuclear protein encoded by p53 gene, which is widely described as the guardian of the genome due to its critical regulatory role during the different phases of cell cycle. The expression of wild-type p53 gene during cell cycle checkpoints helps to regulate DNA repair and apoptosis such that division of genetically impaired cells is arrested to allow repair of genomic alterations and when the DNA repair machinery fails, p53 expression stimulates elimination via apoptosis of impaired cells.[15],[16] As noted earlier, the critical metabolic role exposes the liver tissue to a variety of synthesized or ingested macromolecules. These include phytochemicals of medicinal plants widely used for diverse pharmacological applications. One of such medicinal plant is Ageratum conyzoides Linnaeus which has numerous biological and pharmacological activities. These include treatment of stomach ailments, gynecological diseases, and leprosy, and is used as an anticoagulating and analgesic agent.[17],[18],[19] Its documented biological activities include antioxidant, anti-inflammatory, and antiproliferative activities.[20],[21],[22] These are primarily a function of bioactive phytochemical constituents of the plant such as flavonoids, chromene, coumarin, benzofuran, monoterpenes, sterols, and alkaloids.[23] In this study, the aim was to assess the antiproliferative potential of methanolic extracts of A. conyzoides Linn. using molecular markers such as cell proliferation marker (Ki-67 protein) and tumor suppressor marker (p53 proteins) in the hepatic tissue of adult male Wistar rats following treatment with the extracts.


  Materials and Methods Top


Plant materials

Fresh whole plant of A. conyzoides L. was identified at the Herbarium of Department of Plant Biology and Biotechnology, University of Benin, Nigeria, and documented with a voucher number 344. The plant was sourced from Isihor Community of Benin City, Edo State, Nigeria.

Extract preparation

The leaves of the plant were detached, air-dried, and pulverized into powdered form using a mechanical grinder. 1000 g of the powdered leaves was dissolved in 5 L of methanol for 72 h, was filtered using Whatman's filter paper, and was evaporated to dryness using a rotary evaporator.[24] The residue was allowed to cool, was weighed, and was used as the methanolic extract used for this study.

Experimental animals

This study involved twenty adult male Wistar rats weighing between 170 and 180 g and grouped into five groups (1–4) comprising five animals per group (n = 5). Group 1 was given distilled water (5 ml/kg body weight). This group represented nontreated and noninduced control. Group 2 animals were given 100 mg/kg methanolic extracts of A. conyzoides L, Group 3 animals were given 300 mg/kg methanolic extracts of A. conyzoides L, and Group 4 animals were given 500 mg/kg methanolic extracts of A. conyzoides L. The selected dosages of extracts for this study were considered safe without toxic effects.[25] Administration in all the groups was done orally using a flexible orogastric gavage, and the treatment period of this study was 28 days. Thereafter, the experimental animals were sacrificed and their hepatic tissues were harvested and prepared for tissue processing. All procedures in this study conformed to the International Standard for Experimental Animal Handling and guidelines by the Research and Ethical Committee of College of Medicine, University of Benin, Nigeria.

Tissue processing

The hepatic tissues were fixed in 10% neutral buffered formalin, were dehydrated using ascending grades of alcohol (two changes each of 70% and 90% and absolute alcohol for 30 min each), were cleared in xylene for 30 min, and were embedded in molten paraffin and allowed to cool to form tissue blocks.

Sectioning and staining

Blocks of tissue were cut into sections at 3- and 5-μ thickness by using a rotary microtome and mounted on microscope slides. The 5 μ-thick sections were used for the hematoxylin and eosin staining technique, whereas the 3 μ-thick sections were used for the horseradich-peroxidise-3, 3-Diaminobenzidine (HRP-DAB) staining technique for Ki-67 and p53 proteins using monoclonal antibody.

Hematoxylin and eosin staining technique

The tissue sections were dewaxed in xylene for 15 min, were hydrated with 100%, 90%, and 70% alcohol and distilled water for 3 min each, were stained in hematoxylin for 10 min, were washed under running water for 2 min, were differentiated in 1% acid alcohol (1% HCl in 70% alcohol) for 1 min, blued in Scott's tap water for 10 min, were rinsed in water, were stained in eosin for 3 min, were rinsed in water, were dehydrated with 70%, 90%, and 100% alcohol for 2 min each, were cleared in xylene for 2 min, and were mounted with distrene polystyrene xylene (DPX).[26]

Horseradich-peroxidise-3, 3-Diaminobenzidine immunostaining technique

The sections were brought to water, and antigen retrieval was performed by using citric acid solution (pH 6.0) in a microwave at power 100 W for 15 min. The sections were equilibrated by gently displacing hot citric acid with running tap water for 3 min, were endogenous peroxidase blocked using peroxidase block for 15 min, were washed in phosphate-buffered saline (PBS) mixed with Tween 20 for 3 min, were protein blocked with Nevocastra protein block for 15 min, were washed with PBS for 3 min, were incubated in primary antibody (1 in 100 dilution ratio) for 45 min, were washed with PBS for 3 min, were added secondary antibody and allowed for 15 min, were washed with PBS for 3 min, were added polymer and allowed for 15 min, were washed twice with PBS and treated with DAB substrate for 5 min each (DAB was prepared in 1/100 dilution ratio with the DAB substrate), were washed with water and counterstained with hematoxylin for 2 min, were washed in water, were dehydrated in alcohol, were cleared in xylene, and were mounted with DPX.[27]

Photomicrography

Photomicrographs were generated from stained microscope slides using 10 MP digital camera for microscope. All photomicrographs were analyzed using the Image-J software (National Institues of Health, Bethesda, Maryland, USA) to quantify mucous cell population in periodic acid-Schiff sections and distribution of anti-Ki-67 and anti-p53 in HRP-DAB sections. All values obtained were recorded.

Statistical analysis

Data obtained were analyzed using IBM-SPSS (version 20, IBM Corp., NY, USA) and presented as mean ± standard error of mean. Relevant statistical values were derived using t- test and one-way analysis of variance (P < 0.0010.05 was considered statistically significant).


  Results Top


Histological results

The histological results of this study revealed the histoarchitecture of the hepatic tissues of experimental animals in nontreated Group 1 and treated Groups 2–4, showing densely packed hepatic parenchyma and prominent central vein [Figure 1].
Figure 1: Photomicrograph of histological sections of hepatic tissue of nontreated Group 1 and treated Groups 2–4 showing normal relatively histomorphology with densely packed parenchyma and prominent central vein (H and E, ×100). 1: nontreated group; 2–4: treated groups administered with 100, 300, and 500 mg/kg methanolic extracts of Ageratum conyzoides L., respectively

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Immunohistochemical results

The immunohistochemical results revealed the distribution of cell proliferation (anti-Ki-67) protein and tumor suppressor (anti-p53) protein in hepatic tissues of nontreated Group 1 and treated Groups 2–4, as indicated by the typical brownish staining intensity [Figure 2] and [Figure 3]. The distribution for anti-Ki67 was moderate for all groups except in Group 4 with statistically significantly (P < 0.05) intense staining.
Figure 2: Photomicrograph of immunostained hepatic tissue section of nontreated Group 1 and treated Groups 2–4 showing Ki-67 protein distribution in the hepatic parenchyma with characteristic dark-brown staining product of horseradish peroxidase-3, 3-Diaminobenzidine reaction (×100). 1: nontreated group; 2–4: treated groups administered with 100, 300, and 500 mg/kg methanolic extracts of Ageratum conyzoides L., respectively

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Figure 3: Photomicrograph of immunostained hepatic tissue section of nontreated Group 1 and treated Groups 2–4 showing p53 protein distribution in the hepatic parenchyma with characteristic dark-brown staining product of horseradish peroxidase-3, 3-Diaminobenzidine reaction (×100). 1: nontreated group; 2–4: treated groups administered with 100, 300, and 500 mg/kg methanolic extracts of Ageratum conyzoides L., respectively

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  Discussion Top


The cell proliferation (Ki-67) and tumor suppressor (p53) proteins are among the major markers of cell proliferation that mainly play opposing roles during the process of cell division and proliferation. The expression of wild-type Ki-67 is characteristic of all phases of the cycle of an actively dividing cell.[28] On the other hand, the expression of wild-type p53 protein is most prominent during cell cycle checkpoints, wherein DNA structural integrity is to be maintained or restored via repair machinery without which cell proliferation becomes inhibited via promotion of apoptosis of the genetically impaired cells.[29] In this study, the histological profile of hepatic tissue of the experimental animals revealed densely packed sheets of hepatocytes, most significantly in treated Group 4. In a similar fashion, the immunohistochemical staining result revealed that there is statistically significant (P < 0.05) upregulation of the Ki-67 expression only among the treated Group 4 animals [Table 1] and [Figure 4]. This implies that only higher dose (500 mg/kg) of methanolic extracts of A. conyzoides L. may trigger an increased rate of hepatic cell proliferation. The immunohistochemical staining result further showed that the p53 protein expression was statistically significantly (P < 0.05) upregulated in all treated Groups 2–4 relative to the nontreated Group 1 [Table 2] and [Figure 5], which implied inhibition to hepatic proliferation process following exposure to the methanolic extracts of A. conyzoides L. This proliferative inhibition or antiproliferative activity of A. conyzoides L. has been reported in a study by Adebayo et al.[19] In their study, it was posited that ethanolic extracts of A. conyzoides L. exhibited anticancer activity, and one of the mechanisms involved is its inhibitory effect on cell proliferation. Another study also reported the antiproliferative activity of essential oil derived from selected medicinal plants including A. conyzoides L.[22] From the result of their study, essential oils from A. conyzoides L. exhibited significant antiproliferative activity as well as antioxidant and anti-inflammatory properties. Therefore, according to this study, treatment with methanolic extracts of A. conyzoides L. may downregulate hepatic tissue proliferation, and only higher dosage exposure may help to counteract this antiproliferative influence.
Table 1: Mean and standard error of mean of anti-Ki-67 distribution in hepatic tissue of nontreated Group 1 and treated Groups 2-4

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Figure 4: Chart showing the mean Ki-67 distribution in hepatic tissues of nontreated group (1) and treated groups (2–4). *P < 0.05 was statistically significant when compared to normal control

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Table 2: Mean and standard error of mean of anti-p53 distribution in hepatic tissue of nontreated Group 1 and treated Groups 2-4

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Figure 5: Chart showing the mean p53 distribution in hepatic tissues of nontreated group (1) and treated groups (2–4). *P < 0.05 was statistically significant when compared to normal control

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  Conclusions Top


Based on the dose-dependent and dose-independent expression, respectively, exhibited by the cell proliferation (Ki-67) and tumor suppressor (p53) proteins following exposure to methanolic extracts of A. conyzoides L., the extracts can be said to largely exhibit antiproliferative potential.

Acknowledgments

We acknowledge the contributions of Mrs. Okoro, Histopathology Laboratory, University of Benin Teaching Hospital, Benin City, Edo State, Nigeria, toward the successful completion of this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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