Efficacy of Estrogens and Progesterone in Hepatic Fibrosuppression

By LibertyforLivers

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  • 1Short Project Description 
  • 2Summary 
  • 3About MeAbout Our Team 
  • 4Question / Proposal 
  • 5Research 
  • 6Method / Testing and Redesign 
  • 7Results 
  • 8Conclusion / Report 
  • 9Bibliography, References and Acknowledgements 

Our project explores regressive properties of progesterone, estriol, and estrone in hepatic fibrosis, a scarring process caused by liver inflammation. Prior research has shown these female sex hormones may have preventive effects, but has not established their regressive properties. This project aims to find the hormone most potent in its regressive properties in rat hepatic stellate cell (HSC) cultures, to obtain the lowest effective dosage and hence, reduce side effects of therapy. We treated activated HSC-T6 cells with hormones, and quantified fibrotic markers to to determine treatment efficacy. Our research could provide insight on treatment modalities for potential estrogen-deficient patients.

http://www.youtube.com/watch?v=lM5UruOKtTk

Liver fibrosis is a scarring process caused by liver inflammation which may lead to liver failure. On activation, hepatic stellate cells (HSCs) transdifferentiate into collagen-producing myofibroblasts, causing liver dysfunctionality. Differing rates of prevalence of hepatic fibrosis between males and females has been attributed to anti-fibrogenic effects of estrogens. Estradiol has been shown to have anti-fibrogenic effects by attenuating hepatic stellate cell (HSC) proliferation and activation. It is thus proposed that female sex hormones can be used to induce the regression of hepatic fibrosis. Studies have been focusing on the preventive effect of female sex hormones by pre-treating then inducing hepatic fibrosis. This project was conducted to explore their potential regressive effects by treating HSCs with female sex hormones after activation. It aims to find the most fibrosuppressive hormone at its specific effective, non-toxic dosage to provide the lowest effective dosage and reduce side effects of hormonal therapy and investigate roles of other estrogens (estriol, estrone) and progesterone in suppressing hepatic fibrosis. Activated HSCs were treated with estrone, estriol and progesterone at recommended effective dosages for 48 hours. Cell culture supernatants and lysates were assayed using ELISA and Western Blot to determine fibrotic marker levels such as TGF-β1, TGF-β Receptor I, soluble type I collagen and α-SMA to determine the effects of the hormones on hepatic fibrosis progression. All 3 hormones resulted in fibrotic marker level reductions. Estrone (treated at 200pg/ml) and estriol (treated at 50fg/ml) exhibited the most potent anti-fibrogenic effects, having potential for development into hepatic fibrosis treatments.

We, Yi Xi, Samantha and Tricia, are aspiring medical researchers who study in Raffles Girls' School (Secondary) in Singapore. After taking part in an international biomedical olympiad, we learnt that medical research is instrumental in curing the complex diseases which occur in the human body. Our passion for the pursuit of scientific inquiry has led us to initiate a project on liver fibrosis. We decided to participate in the Google Science Fair as we hope it can provide a platform for us to share our discoveries with the world. A key scientist who has inspired us is Professor Hanry Yu, who mentored us during our research journey. Being an expert in the study of hepatic fibrosis, he offered us invaluable guidance. As budding scientific researchers, we looked up to him as a role model due to his dedication to and passion for his work.  In the future, we hope to delve deeper into the field of biomedical research and further add to the existing pool of scientific knowledge. We hope that our contributions to scientific research can be used in real-life applications to improve patient welfare, as we believe each scientist has a duty to work towards the betterment of society. Winning this competition would go a long way in empowering us to further our studies, which will give us a greater capacity to contribute to improving society's well-being through our research endeavors, and allow us to repay the efforts of Professor Yu and our school in nurturing us.

This project aims to find the hormone which is most potent in its fibrosuppressive properties at its specific effective, non-toxic dosage according to literature so as to provide the lowest effective dosage and concurrently reduce the negative side effects of hormonal therapy.

It is hypothesized that estrogens and progesterone play significant roles as fibrosuppressants. The hypothesis of the most effective fibrosuppressant is based on key factors, such as the hormone’s potency. Based on the above, progesterone is hypothesized to be the most effective, followed by estrone and then estriol. Estriol is supposedly the least effective as it is the weakest out of all 3 hormones. Estrone is hypothesized to be the next least effective, for despite its anti-inflammatory and anti-oxidant properties, there are conflicting studies on its fibrosuppresive properties. Progesterone shows potential as being the most effective fibrosuppressant due to fewer conflicting studies, and its potency shown in prior applications in hormonal therapies.

Serum type I collagen levels are positively correlated with hepatic fibrosis [14]. Hence, TGF-β1 and type I collagen will serve as markers for the severity of fibrosis. Moreover, levels of α-SMA, a marker for activated HSCs [15], and TGF-β Receptor I (TGF-βRI) [16], a downstream marker for fibrosis whose levels positively correlate with TGF-β1 levels, will also be measured.

The amount of fibrotic marker in each treatment will be compared against the control.  The lower the fibrotic marker levels in the cell lysate and supernatant, and the lower the treatment dosage, the more fibrosuppressive the hormone.

Liver fibrosis, a scarring process caused by liver inflammation, may eventually lead to cirrhosis or liver failure. On activation, hepatic stellate cells (HSCs) transdifferentiate into collagen-producing myofibroblasts, causing liver dysfunctionality.

 

Studies have demonstrated that fibrosis is more common in men than in women [1] [2]. The ratio of male:female patients has been reported to range from 2.3:1 to 2.6:1 in hepatic fibrosis and cirrhosis [3]. It has thus been proposed that sex hormones may play significant roles in the pathogenesis of fibrotic diseases. It has been hypothesized that the estrogens may play an important role as endogenous fibrosuppressants, and a study done on both male and female rat livers has shown the presence of high affinity, low capacity estrogen receptors (ER) that respond to estrogens by regulating liver function [4]. Recent studies have been focusing on the preventive effect of female sex hormones on hepatic fibrosis by pre-treating, then inducing hepatic fibrosis [5]. This project aims to explore regressive effects of female sex hormones on hepatic fibrosis, by treating hepatic stellate cells (HSCs) with female sex hormones after activation.

 

Further studies have shown that estradiol, the most potent and predominant female sex hormone causes an increase in hepatic ERs and their mRNA, [6] and attenuates the activation and proliferation of cultured rat HSCs, which are responsible for most of the collagen deposition in hepatic fibrosis [7]. Estradiol treatment also suppressed hepatic collagen content and reduced hepatic type I collagen in rats with CCl4-induced fibrosis [8]. However, the precise role of other types of estrogens (estriol and estrone) and progesterone, another steroid hormone present in females, in the suppression of hepatic fibrosis has not been well established. The effects of these hormones on hepatic fibrosis are of interest as they are predominant at different stages of a woman’s life cycle, which may lead to differing susceptibility to hepatic fibrosis.

 

The present study was thus initiated to investigate and compare the effects of estrone, estriol and progesterone on TGF-β1 and type I collagen expression in rat HSC cultures. TGF-β1 is a profibrotic cytokine involved in hepatic fibrogenesis [9]. Activation of TGF-β1 leads to transdifferentiation of HSCs into myofibroblasts, and stimulates the synthesis of extracellular matrix proteins while inhibiting the degradation of these proteins [10]. Prior studies have shown a correlation between patients with fibrosis and elevated levels of TGF-β1. [11] TGF-β1 expression also increases type I procollagen mRNA levels [12] and causes accumulation of collagen through myofibroblast transdifferentiation [13]. Levels of serum type I collagen are positively correlated with hepatic fibrosis [14]. Hence, TGF-β1 and type I collagen will serve as markers for the severity of fibrosis. Moreover, levels of α-SMA, a marker for activated HSCs [15], and TGF-β Receptor I (TGF-βRI) [16], a downstream marker for fibrosis whose levels positively correlate with TGF-β1 levels, were also measured.

Cell Culture

The cell culture is an immortalised HSC-T6 monoculture system immersed in DMEM Media (with red phenol indicator with L-Glutamine). HSC-T6 cells were cultured in DMEM with 10% serum before treatment.

Hormone Treatment

Powdered hormones (Sigma) were diluted in dimethyl sulfoxide (DMSO; Riedel-de Haen (60153)) to form the stock concentration, which was diluted in media without serum (0.2% DMSO). 0.2% DMSO is the suggested concentration used in existing studies to avoid cellular toxicity.

There are 5x2 wells with duplicates, two wells each for a negative control treated with DMEM media, a negative control treated with 0.2% DMSO, progesterone, estrone and estriol. HSC-T6 cells were seeded at 1x10^5/ml for 2ml per well in 6-well plates (NUNC) and at the same concentration for 500 µl per well in the 24-well plate (NUNC). Two days after seeding, media was changed and media without serum, with hormone dosages was added. Treatment dosages are as follows: progesterone (600 pg/ml), estriol (50 fg/ml) and estrone (200 pg/ml) . Treatment dosages were obtained from existing literature. Cells were incubated for 48 hours at 37°C, 5% CO2. Supernatant and cell lysate was collected and stored in -20°C freezer. ELISA and Western blot were used to determine the amount of type 1 collagen, TGF-β1, α-SMA and TGF-βRI in cell supernatant and lysate samples. Each setup also consisted of a 24-well plate used for Alamar Blue Assay to determine cell viability.

Protein Quantification

Protein concentrations were determined using standard Bradford Assay kit (Bio-Rad). Known concentrations of TGF-β1 were used to generate a standard graph, beginning from 1ng/ml. Linear fit curve (y=0.0008x) was obtained and used to calculate the unknown protein concentrations.

ELISA – TGF-β1 and Type I Collagen

Cell supernatants and lysates were assayed for active TGF-β1 using TGF-β1 Emax immunoassay kit (Promega). Levels of Type I collagen levels were determined through ELISA. The absorbance of the resulting solution was read using spectrophotometry at 450 nm.

Western Blot - α-SMA, TGF-βRI

Protein measurements using western blot were carried out with cell lysates from 48hr hormone conditioned HSC-T6 monoculture to test for levels of . α-SMA and TGF-βRI. Band intensity was measured using ImageJ (WS Rasband, National Institutes of Health, Bethesda, MD).

Alamar Blue Assay

Alamar Blue Assay was conducted on the 24-well plate once 24 hours before treatment (after seeding), 24 hours after first treatment and 24 hours after second treatment. 50µl Alamar Blue® reagent diluted with 450µl of DMEM media was added to each well. The plates were incubated at 37°C for 2 hours before measuring the fluorescence signal.

Statistical Analysis

The results obtained were from three or more independent set of experiments; the values were expressed as Mean ± SEM and considered significantly different if the p-value < 0.05. The p-value was calculated from two-tailed unpaired student’s t-tests. The Analysis of Variance (ANOVA: Single Factor) from Microsoft Excel (value of Alpha = 0.05) was used to determine that there was no significant difference between the control treated with DMEM media and the control with 0.2% DMSO.

Alamar Blue Assay

Alamar Blue Assay showed no significant difference in cell proliferation between all treatments (DMSO and hormones) and the untreated control. Hence, differences in levels of fibrotic markers cannot be attributed to differential cell death between treatments, but can be attributed to hormonal action on the various fibrotic signalling pathways. 

Comparison of Control with DMSO against Control without

Cell culture supernatant and lysate samples were assayed for active TGF-β1 by ELISA. Three sets of data were analyzed using ANOVA (critical f-value of 7.7086) to determine that there was no significant difference between the untreated control and the control treated with 0.2% DMSO. Any differences in TGF-β1 levels between the control without DMSO and the treated cells can thus be attributed to hormonal action rather than DMSO action, enabling a comparison.

Effects of Hormonal Treatment on Active TGF-β1 levels (ELISA)

Cells treated with progesterone, estrone and estriol exhibited significant decreases in active TGF-β1 levels in supernatants relative to the control, as seen in Figure 1. TGF-β1 in the supernatant can interact with multiple cells to cause profibrotic effects. Hence, hormonal treatment could significantly aid fibrotic regression, especially since TGF-β1 signalling has a prime role in fibrotic progression.

Estrone treatment significantly reduced TGF-β1 levels in cell lysates (Fig. 1). Estrone treatment was the only treatment which consistently lowered TGF-β1 in both cell lysate and supernatant samples, possibly indicative of it having the most potent regressive effects of the hormones investigated.

Effects of Hormonal Treatment on Type I Collagen levels

Supernatants were assayed for type I collagen levels by ELISA to determine how treatment affects type I collagen synthesis. 

Progesterone, estrone and estriol caused small decreases in type I collagen levels as seen in Fig. 2. Despite there being no significant trends in data, decreases in collagen levels were especially prominent in progesterone and estrone treatments. These hormones thus demonstrate potential in effecting fibrotic regression as serum type I collagen levels are positively correlated to the severity of hepatic fibrosis.

Effects of Hormonal Treatment on TGF-βRI and α-SMA Levels (Western Blot)

RIPA fractions (lysate) were assayed for TGF-βRI and α-SMA by Western Blot to determine how treatment affects TGF-β1 signalling and HSC activation repsectively. Results were normalized to β-Actin.

With reference to Fig. 3, Western Blotting showed no significant reduction in TGF-βR1 and α-SMA levels in treated samples relative to the control. As treatment was only done for 2 days, there may have been insufficient time for decreases in TGF-β1 levels to translate to a corresponding decrease in the levels of these markers as both are downstream of TGF-β1 in the TGF-β1 signalling pathway. Moreover, α-SMA is a marker for activated HSCs. Since cells were activated prior to treatment; treatment may have had less discernible effects on α-SMA levels than if cells had been treated pre-activation.

DISCUSSION

Estriol and estrone may have suppressed hepatic fibrosis through estrogen receptors [22]. Estrone seems to be the most effective at reducing both intracellular and extracellular TGF-β1 levels, and is thus the most potent fibrosuppressant of the hormones at these dosages. However, estriol treatment caused reductions in TGF-B1 levels similar to estrone’s despite its lower dosage (4000x lower), and could be better for therapeutic applications as side effects are minimized due to the lower dose. Since the effective dosage is much lower than estrone (4000x) and progesterone (12000x), the difference between the lowest effective dosage and toxic dosage for estriol could be greater, and thus there is more potential for the dosage to be increased and yet kept within safe limits. Treatment with progesterone was less successful at reducing TGF-β1 levels (similar reduction in supernatant concentrations despite its dosage being 3x higher than estrone and 12000x higher than estriol). This could be due to progesterone being more favourable to fibrogenic effects such as TGF-β1 expression, as shown by other studies. It has been proposed that such effects occur due to progeserone's induction of reactive oxygen species generation and intracellular pathways [23].

CONCLUSION

Results  of  statistical  tests  to  ascertain  relationships  between  fibrotic  marker  levels  (Type  I  collagen,  TGF-β1, α-SMA, TGF-βRI) and form of hormone treatment showed that estrone (200pg/ml) is the most potent in lowering the rate of liver fibrotic progression, though estriol demonstrates potential as well considering that it caused similar reductions in TGF-β1 levels in supernatants despite its lower dosage. Due to anti-fibrogenic effects caused by treatment with estriol and estrone, there is potential for these estrogens to be developed into hepatic fibrosis treatments. The results show that female sex hormones have regressive, not just preventive effects in hepatic fibrosis as they can interfere with TGF-β signalling in HSCs at the post-activation stage.

LIMITATIONS OF THE CURRENT STUDY

As the study was conducted on a HSC monoculture and not on live animals, it is unable to predict conditions in vivo. Thus, it does not take into account how hormone treatments could affect other cells that play a role in hepatic fibrosis.

RECOMMENDATIONS FOR FURTHER RESEARCH

The experiment could be repeated to determine if estriol or estrone has more potent fibrosuppressive properties. Cells could be treated with a range of hormone concentrations to identify the lowest effective dose to minimize side effects of hormonal therapy. Knowledge on how treatment effectiveness peaks can aid in maximizing curative potential. Additional markers involved in fibrosis can be investigated, not just in the TGF-β pathway but in other pathways such as PDGF (platelet-derived growth factor), and identify the exact mechanisms which enable estrogens and progesterone to suppress fibrosis within HSCs.

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