Western Herbal Medicine,Epigenetics,and Endometriosis

Abstract

Endometriosis is an enigmatic disease characterized by the presence and growth of endometrial-like tissue outside the uterine cavity. The etiology of endometriosis is poorly understood, yet recent evidence suggests that epigenetic aberrations and heritable changes in the genome may be the key to understanding how to approach this disease. Difficulty in long-term management of endometriosis symptoms and unpredictability of treatment outcome necessitate research into other treatment modalities, such as Western herbal medicine. This article reviews commonly used herbs in the treatment of endometriosis, the effects of phytochemical constituents on endometrial cells, and the impact on the epigenome.

Introduction

A pproximately 10% of Western women of reproductive age are diagnosed with endometriosis.¹ Endometriosis is an estrogen-dependent disease characterized by inflammation and growth of endometrial-like tissue outside the uterine cavity.² The pelvic organs and the peritoneum are the most commonly affected.³ Molecular evidence of progesterone resistance in eutopic endometrium is linked to estrogen dominance, but the complete etiology of endometriosis is not fully understood.⁴ Theories of coelomic metaplasia,⁵ Müllerian remnants,⁶ and implantation from retrograde menstruation⁷ have all been postulated. Retrograde menstruation involves the movement and subsequent implantation of viable endometrial cells into the peritoneum during menstruation.⁷ As a consequence of refluxed endometrial cells, inflammation occurs in the peritoneum.¹ Despite being the most widely accepted theory, retrograde menstruation occurs in 90% of menstruating women, whereas endometriosis only occurs in up to 10% of women of reproductive age,⁸ which brings into question the accuracy of this theory. Furthermore, there is significant variation in the severity of endometriosis, suggesting that several factors may be involved in its pathogenesis.⁹

Current management of endometriosis is based on reduction of pain and prevention of pain recurrence. Pain management options include nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesics; suppression of ovarian function with hormonal drugs (oral contraceptives, danazol, gestrinone, GnRH agonists, dienogest); and surgery (laparoscopic ablation of endometriotic lesions).¹⁰ Yet, there is inconclusive evidence to show pain management with NSAIDs is effective in endometriosis; moreover, significant adverse effects on the gastrointestinal tract and ovulatory disturbances can occur if NSAID use coincides with the middle of the menstrual cycle.³ The hormonal drugs also produce varying levels of side-effects that in many cases prevent long-term use and can lead to poor compliance.³ Progestins aim to suppress growth and inflammation and thus reduce pain through inhibition of ovarian estrogen biosynthesis. Although pharmacological pain relief can work for some women, unfortunately it is often short-term and some women do not respond to therapy at all.¹¹ Recent evidence does show efficacy and tolerability of dienogest, a 19-nortestosterone derivative, for endometriosis treatment.^12
–14 In general, however, lack of outcome prediction associated with pharmacological treatments and the uncertainty over individual response rates highlight a need to investigate other approaches to managing this disease.¹⁰

Documented historical use of herbal medicine for endometriosis-like symptoms is evident in Chinese literature.¹⁵ There is now growing popularity in the West for the use of herbal medicine in treating endometriosis to try to overcome the poor long-term success at resolving chronic pain or recurrence of disease using conventional intervention.^10,16 Although recent data on dienogest for pain management in endometriosis provides a monotherapeutic additional avenue for gynecologists to explore, the Royal College of Obstetricians and Gynaecologists (RCOG) reports that some women with endometriosis choose complementary medicine (including herbal medicine). This may be as a treatment option to avoid hormonal therapy, whereas other women are looking for a multitherapeutic strategy to manage their condition^3,17 and thus combine herbal medicine with prescribed pharmacological treatment.

Common herbs used by women with endometriosis have demonstrated antiproliferative, antioxidant, analgesic, and anti-inflammatory effects on endometrial cells, showing promise in the treatment of endometriosis in vitro. ^15,18 The understanding of herbal clinical practice, however, outside the herbal medicine community is poor,¹⁹ making it difficult to integrate herbal medicine into a multidisciplinary treatment strategy.

Diagnosis of a condition in Western herbal medicine is based on a conventional medical model of pathology.¹⁹ Patients receive an individual herbal formula (often a mixture of two to three herbs) chosen from a wide range of plants and usually given as concentrated liquid (either as a tea or alcoholic tincture).¹⁹ Herbal tablets and capsules are also starting to be prescribed more regularly, although this is a more standardized approach to treatment that is moving away from traditional prescribing.¹⁹ The patient will also receive individual therapeutic advice relating to diet and lifestyle.²⁰ The individual approach to treatment with herbal medicine aims to address the individual needs of each patient and the prescription will be adapted to the changing clinical presentation as the patient moves through treatment.²¹

Evidence exists for the involvement of immunological, hormonal, genetic, and environmental factors in endometriosis, as well as the deregulation of multiple biologic pathways.^22

–27 More recently, epigenetic changes have also been hypothesized as a common denominator in endometriosis.²⁸ The connection between environmental impact and susceptibility to deregulation of gene expression is very interesting. Of more importance to women suffering with endometriosis, however, is the fact that epigenetic changes such as DNA methylation states, regulation of gene expression by microRNA (miRNA), and histone modification appear to be reversible. These states can be moderated by lifestyle and the environment, which creates an unpredictable combination of circumstances underlying the expression of symptoms.²⁹ It is this individuality of disease and complex inheritance patterns of endometriosis that herbal medicine aims to address with its underlying principle of individualized practice.

This article will now examine the contribution of epigenetic changes in gene expression in endometriosis and discuss how known mechanisms of actions of Western herbs commonly used to treat endometriosis relate to existing knowledge of the etiology of endometriosis.

Epigenetics and Endometriosis

Epigenetics relates to changes in chromatin structure and/or gene expression without any mutations in the DNA sequence. Epigenetic effects to be discussed in this article include changes to the methylation status of a promoter region of a gene, changes to the acetylation state of DNA, and modulation of gene expression by miRNAs.

Methylation of promoter regions is the most studied epigenetic molecular mechanism, most often associated with gene silencing (hypermethylation), but sometimes with increased gene expression (hypomethylation).^30,31 Aberrant methylation in human endometriotic cells has been suggested as a biologically plausible mechanism for changes in gene expression of progesterone receptor B (PR-B) and estrogen receptor-ß (ER-ß).³² The promoter region of PR-B but not progesterone receptor A (PR-A) showed evidence of hypermethylation in endometriotic implants taken from human tissue biopsies.³³ Furthermore, stimulation of an epithelial-like immortalized endometriotic cell line (11Z), with tumor necrosis factor-α (TNF-α), induced PR-B hypermethylation.³⁴ This suggests that a chronic inflammatory disease state may lead to PR-B promoter hypermethylation and therefore progesterone resistance in endometriosis.³⁴ Hypomethylation of the promoter region of ER-ß and subsequent increased expression of ER-ß in endometriotic stromal cells taken from human tissue biopsies compared with normal endometrial cells has also been reported.³⁵ High levels of ER-ß suppress estrogen receptor-α (ER-α) expression, a certain level of which is needed for estradiol induction of the PR promoter region. Hence, hypomethylation of the ER-ß promoter may further induce progesterone deficiency in endometriosis.³⁵ The progesterone resistance due to reduced expression of PR-B and suppression of ER-α may be a possible explanation for the poor clinical response to progestins.³⁴

Homeobox protein HOX-A10 is encoded by the HOX-A10 gene, which is part of a family of genes that serve as transcription factors during embryo development. HOX-A10 expression mediated by sex steroids has been investigated for its role in uterine function and embryo implantation.³⁶ A significant reduction in HOX-A10 expression normally expected in the midsecretory phase of the menstrual cycle is shown in women with endometriosis, suggesting defective uterine receptivity and the potential for reduced fertility.³⁷ A suggested reason for this finding is due to partial hypermethylation of the HOX-A10 gene and therefore reduced expression of HOX-A10 in the endometrium.³²

Understanding of the molecular mechanisms underlying methylation changes still is not clear; however, overexpression of the enzymes that catalyze DNA methylation may be implicated.³⁸ DNA methyltransferases (DNMTs)—DNMT1, DNMT3A, and DNMT3B—are all overexpressed in the epithelial component of endometriotic tissue taken from women with endometriosis as compared to control tissue.³⁹ The heritable and persistent nature of endometriosis indicates there must be a mechanism maintaining aberrant changes. DNA methylation shown through PR-B, ER, HOX-A10, and DNMTs changes may be that mechanism, indicating that endometriosis could be an epigenetic disease.³⁹

Another aspect of epigenetics relates to miRNAs, which are short ribonucleic acid molecules that are present in all human cells and that regulate up to 30% of human genes,⁴⁰ through inhibition or enhancement of mRNA translation and induction of protein degradation.^28,41 Studies have shown downregulation of 48 of 487 miRNAs in endometriotic lesions.⁴² Of those 48 miRNAs identified, their target genes included the following: transforming growth factor-ß, ER-α, ER-ß, and PR, and changes in expression of all of these genes have been linked to endometriosis. Modulation of gene expression via miRNAs may therefore add an additional layer of gene regulation.²⁸ Other studies looking at miRNA functions in endometriosis have shown evidence for their influence on inflammation, tissue repair, cell growth, cell proliferation, apoptosis, and angiogenesis.^43
–45 For example, miR-199a and miR-16 that suppress cyclooxygenase-2 (COX-2) translation show down-regulation in endometriosis, hence increasing COX-2 levels and inflammation,⁴² whereas an increased expression of miR-15b/16, miR-145, and downregulation of miR-20a, miR-221, and miR-222 have been linked to increased survival of endometriotic cells.⁴⁶ It is worth noting, however, that differences have been seen between the lists of miRNAs that have been identified and their direction of expression, possibly due to variations between individuals.⁴⁷

Endometriosis Signaling Pathways and Phytochemical Agents

Table 1 shows the most commonly used Western herbs that are prescribed for women with endometriosis at the University of Westminster's polyclinic. As noted in Table 1, many herbs have known properties that are anti-inflammatory, anti-proliferative, analgesic, antispasmodic, and antioxidant, which will now be discussed further.

Table 1.

Summary of the Pharmacological Effects of Herbal Medicines and Their Role in the Treatment of Endometriosis

		Anti-inflammatory
Herbal medicine	Antioxidant	Inhibition of NF-_kB	Inhibition of leukotrienes	Inhibition of prostaglandins	Inhibition of peritoneal macrophages	Inhibition of cytokines	Anti-spasmodic	Anti-proliferative/apoptotic	Anti-nociceptive/analgesic
Angelica sinensis ^52,99	^*	^*		^*		^*
Glycyrrhiza glabra ⁸⁷	^*			^*	^*
Phytolacca Americana ^60,97								^*	^*
Centella asiatica ⁶¹	^*							^*
Calendula officinalis ^{62,88,100,101}	^*			^*			^*	^*
Viburnum prunifolium ¹⁰²							^*
Chelidonium majus ^{96,100 –103}						^*	^*	^*	^*
Zingiber officinalis ^54,55,89	^*			^*			^*		^*
Achillea millefolium ^64,103		^*						^*
Alchemilla vulgaris ⁶⁵	^*		^*					^*
Thuja occidentalis ^66,67								^*
Carduus marianus ^51,68,90	^*	^*				^*		^*
Hyoscyamus niger ^98,104							^*		^*
Curcuma longa ^56,73,89	^*	^*				^*		^*
Schizandra chinensis ⁹¹	^*		^*

Mechanism unclear.

The local inflammation response in endometriotic tissue encourages cell proliferation, evasion of immune surveillance, and local neuroangiogenesis.¹ More specifically, the activation of macrophages, proinflammatory expression of cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12) and TNF-α, and long-term activation of the innate immune system regulating intracellular signaling pathways through nuclear factor-kB (NF-kB), is linked to cell survival in endometriosis.^48
–50 NF-kB also upregulates COX-2 transcription, which is a pro-inflammatory and pro-angiogenic mediator, increasing estradiol production by enhancing aromatase activity and so contributing to cellular proliferation and continued development of endometriotic lesions.^23,46 Interestingly, there are a few Western herbs that are known to act on aspects of these inflammation associated pathways. For instance, phytochemical constituents from Carduus marianus, Angelica sinensis, and Curcuma longa have demonstrated in vitro and in vivo inhibition of NF-kB, in mouse CD4+ splenocytes⁵¹; lipopolysaccharide (LPS)-stimulated RAW264.7macrophages⁵²; and endometriotic stromal cells,⁵³ respectively. Curcumin the active component of Curcuma longa inhibits secretion of IL-6 and IL-8, reducing inflammation, as well as inhibiting mRNA expression of intercellular and vascular cell adhesion molecules in endometriotic stromal cells through suppression of TNF-α.⁵³

Prostaglandin E2 (PGE2) is one of the prostaglandins synthesized from arachidonic acid by COX-1 and 2. It is secreted in response to inflammation and acts through autocrine or paracrine signaling. PGE2 induction of aromatase mediated by increased intracellular levels of cyclic adenosine monophosphate (cAMP) occurs in endometriotic tissue but not in healthy uterine tissue.²⁴ Subsequent increased local estradiol concentration as a result of increased aromatase expression will also increase COX-2, leading to inflammation and a positive feedback cycle.²⁴ Ligustilide (a constituent of Angelica sinensis), [6]-shogaol and [6]-gingerol (constituents of Zingiber officinalis), and glabridin and isoliquiritigenin (constituents of Glycyrrhiza glabra), are able to inhibit PGE₂ and COX expression, in LPS-stimulated RAW264.7 macrophages^52,54; mouse skin⁵⁵; and LPS-induced PGE2 release in murine macrophages (J774A.1),⁵⁶ respectively.

The COX-2/PGE2 signaling pathway might also suppress apoptosis via activation of other pathways including the Ras-MAPK/ERK pathway, PI3K/AKT pathway, ERK signaling, cAMP/protein kinase A signaling, and the nuclear peroxisome proliferator-activated receptor-δ, as shown in colorectal carcinoma cells.⁵⁷ Insensitivity to antiproliferative signals via progesterone⁵⁸ and cyclin-dependent kinase inhibitors⁵⁹ is also evident in endometriosis. Interestingly, extracts and phytochemical constituents of several herbs listed in Table 1 (Phytolacca americana,⁶⁰ Centella asiatica,⁶¹ Calendula officinalis,⁶² Chelidonium majus,⁶³ Achillea millefolium,⁶⁴ Alchemilla vulgaris,⁶⁵ Thuja occidentalis,^66,67 Carduus marianus,^68,69 and Curcuma longa ^70,71) show antiproliferative effects when tested in human cancer cell lines and mouse models. This demonstrates an alternative antiproliferative signaling pathway to that mediated by progesterone.

Changes in DNA methylation patterns are increasingly being linked to chronic inflammation.^72
–74 For example, epigenetic silencing of suppressor of cytokine signaling 3 (SOCS3) through hypermethylation of the SOCS3 promoter is responsible for sustained inflammation via IL-6 signaling.⁷⁵ Another study has shown that DNA damage from halogenation of nucleic acids in areas of tissue inflammation produces methylation changes and the activation of genes involved in cell proliferation.⁷⁴ Furthermore, abnormal DNA hypomethylation of the nuclear factor for IL-6 site within the promoter of the COX-2 gene in eutopic endometrium of endometriosis has been evidenced, providing a mechanism for upregulated COX-2 expression.⁷⁶

Disruption of the acetylation/deacetylation balance has been implicated in chronic inflammation, leading to sustained transcription of proinflammatory genes such as NF-kB.⁷⁷ Histone modifications such as deacetylation (gene silencing) and histone acetylation (gene expression) have also been reported in endometriosis studies. In vitro studies looking at the effects of histone deacetylase inhibition (HDAC-i) on immortalized endometriotic cell lines and endometriotic cyst stromal cells showed acetylation of histone proteins, preventing proliferation and increasing apoptosis, suggesting that endometriosis is sensitive to epigenetic modifications through histone deacetylase activity.^78
–80

Inflammatory cellular activity and free iron found in endometriotic cysts producing reactive oxygen species in the peritoneal cavity can also contribute to DNA, histone, and chromatin damage and affect gene activation.^74,81
–83 NF-kB is redox sensitive and can be activated through increased intrinsic histone acetyltransferase activity and simultaneous inhibition of HDAC activity.⁸⁴ The result of this is a chronic inflammatory response due to increased inflammatory gene expression. Thiol molecules and dietary polyphenols have been shown to modulate HDAC activity through antioxidant and anti-inflammatory effects, and so altering pro-inflammatory gene activation.⁸⁴ Supplementation of N-acetyl-l-cysteine, a cellular precursor to glutathione, has been shown to downregulate NF-kB in endothelial cells.⁸⁵ C. longa (turmeric) also increases antioxidant glutathione levels through induction of γ-glutamylcysteine synthetase, which is the first enzyme of the glutathione biosynthesis pathway.⁸⁶ Phytochemical constituents and extracts of Angelica sinensis,⁵² C. asiatica,⁶¹ A. vulgaris,⁶⁵ Glycyrrhiza glabra,⁸⁷ C. officinalis,⁸⁸ Zingiber officinale,⁸⁹ C. marianus,⁹⁰ and Schizandra chinensis ⁹¹ also all show antioxidant activity in studies.

Few studies have investigated the therapeutic use of antioxidants in endometriosis, but some studies have shown positive results in reducing pelvic pain and inflammation.^92,93 The last section of this article will discuss some aspects of pain in endometriosis that may be modulated by herbal constituents.

Nerve invasion by endometriotic cells can stimulate the sensory and sympathetic nerves, exerting an effect on the neurons in the central nervous system and increasing pain perception.¹ Changes in nociceptive pathways in the spinal cord and central nervous system can alter gene transcription. However, although epigenetic alterations are implicated in chronic pain, and persistent inflammatory and neuropathic pain, it is unclear how these epigenetic mechanisms operate in the development of these pain states.^94,95 Chronic inflammatory and neuropathic pain states appear to downregulate the transcription of enzyme glutamatic acid decarboxylase in rat brainstem neurons, which impairs the synaptic inhibition of γ-aminobutyric acid (GABA). Treating with HDAC restored GABA function and reduced the pain sensitivity,⁹⁵ suggesting that epigenetic modification of glutamatic acid decarboxylase was related to pain symptoms.

An aqueous extract of Chelidonium majus—a herb with analgesic properties, commonly used in the treatment of endometriosis—was able to suppress GABA and elevate glutamate-activated ion channels in rat periaqueductal gray neurons, which are part of the pain control system.⁹⁶ This may indicate a use for this herb in chronic pain conditions such as endometriosis. Antinociceptive properties similar to morphine were also shown in mice using the aerial parts of Phytolacca americana,⁹⁷ and Hyoscyamus niger seeds showed analgesic activity in mouse models with a possible mechanism of action through the central and peripheral nervous system.⁹⁸

Conclusions

The unpredictable response to conventional pharmacological intervention in some women with endometriosis has led to an increase in the number of patients turning to herbal medicine. This article has postulated that the benefit of several herbs used to treat endometriosis may be attributed to known effects on the signaling pathways associated with the pathogenesis of endometriosis, hence demonstrating antiproliferative, antioxidant, analgesic, and anti-inflammatory effects on endometrial cells.^15,18 Furthermore, the epigenetic processes described are dynamic, and it is likely that use of specific herbs may offer therapeutic interventions to potentially reverse the aberrant epigenetic changes seen in endometriosis. Research shows evidence of these phytochemical constituents positively impacting the epigenome, indicating a potential role for herbal medicine alongside conventional medicine for the treatment of endometriosis.

It must be recognized that the existing research on the cited medicinal plants and their therapeutic use is limited and in many cases can only be based on in vitro studies and animal models. There is nevertheless convincing evidence of the potential of these herbs in endometriosis treatment. Future research needs to establish the pharmacological evidence for the phytochemical effects at a cellular level, and to investigate the efficacy and effectiveness of the herbal combinations and therapeutic strategies used in current herbal practice.

Footnotes

Disclosure Statement

No competing financial interests exist.

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