The Impact of Genistein Supplementation on Tendon Functional Properties and Gene Expression in Estrogen-Deficient Rats

Abstract

Tendinopathy risk increases with menopause. The phytoestrogen genistein prevents collagen loss during estrogen deficiency (ovariectomy [OVX]). The influence of genistein on tendon function and extracellular matrix (ECM) regulation is not well known. We determined the impact of genistein on tendon function and the expression of several genes important for the regulation of tendon ECM. Eight-week-old rats (n = 42) were divided into three groups: intact, OVX, or OVX-genistein (6 mg/kg/day) for 6 weeks. Tail fascicles were assessed with a Deben tensile stage. Achilles tendon mRNA expression was determined with digital droplet polymerase chain reaction. Compared to intact, fascicle stress tended to be lower in untreated OVX rats (P = .022). Furthermore, fascicle modulus and energy density were greater in genistein-treated rats (P < .05) compared to intact. Neither OVX nor genistein altered expression of Col1a1, Col3a1, Casp3, Casp8, Mmp1a, Mmp2, or Mmp9 (P > .05). Compared to intact, Tnmd and Esr1 expression were greater and Pcna and Timp1 expression were lower in OVX rats (P < .05). Genistein treatment returned Tnmd, Pcna, and Timp1 to levels of intact-vehicle (P < .05), but did not alter Scx or Esr1 (P > .05). Several β-catenin/Wnt signaling-related molecules were not altered by OVX or genistein (P > .05). Our findings demonstrate that genistein improves tendon function in estrogen-deficient rats. The effect of genistein in vivo was predominately on genes related to cell proliferation rather than collagen remodeling.

Introduction

The transition through menopause is a significant risk factor for tendinopathies (tendon pain/degeneration), an understudied clinical problem.¹ Menopause also leads to deterioration in tendon function,² changes that likely contribute to the global decline in musculoskeletal function, and reduced quality of life associated with menopause.^3,4 Estrogen appears to be vital for maintenance of tendon extracellular matrix (ECM).^5–9 Specifically, we have shown that in a rat model of estrogen deficiency (ovariectomy [OVX]), estrogen loss results in a large decline in tendon collagen content,⁹ the primary structural protein of tendon. Furthermore, in tendon cell culture, estrogen deficiency results in decreased cell proliferation, reduced type I collagen, and altered tendon metabolism and healing, more than attributable to aging alone.¹⁰ Tissue collagen content directly correlates with tissue stiffness,¹¹ implying that a reduction in tendon collagen could contribute to a reduction in tendon functional properties. However, the impact of estrogen loss on tendon functional properties has not been extensively studied.¹² Furthermore, the molecular mechanisms modulating the effects of estrogen on tendon are not well defined.

A common therapeutic choice for estrogen deficiency, primarily postmenopause, is estrogen therapy (ET). However, in postmenopausal women, ET is associated with tendons that have a lower Young's modulus and a greater number of smaller collagen fibrils.⁶ ET also limits tendon hypertrophy with exercise in postmenopausal women.^5,13 Moreover, it has been suggested that tendinopathy is more common with ET compared to no ET.¹⁴ Collectively, these findings suggest that alternative approaches to minimizing the impact of estrogen loss on tendon are needed.

Genistein, a plant-derived, naturally occurring isoflavone phytoestrogen found in soy, may be a useful strategy to reduce the effects of estrogen deficiency on tendons.⁹ Genistein is thought to convey many of the health benefits associated with a diet high in soy.^15–19 The beneficial effects of genistein on bone and cardiovascular disease remain controversial,²⁰ but in an OVX rat model, we demonstrated that genistein reverses the decline in tendon collagen associated with the loss of estrogen.⁹ However, it is unclear if genistein can improve tendon mechanical properties in estrogen-deficient rats. The objectives of this investigation were to (1) determine if genistein can improve tendon functional properties in estrogen-deficient rats and (2) examine the impact of estrogen loss and genistein supplementation on key regulators of ECM that could account for the changes in tendon collagen noted in our previous work.⁹

Materials and Methods

Study protocol

Eight-week-old female Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA, USA) as either OVX (n = 28) or intact (n = 14). OVX rats were randomly assigned to one of two groups: OVX-vehicle or OVX-genistein. OVX rats received a daily subcutaneous injection of either 6 mg/kg/day of genistein (LC Laboratories, Woburn, MA, USA) in dimethyl sulfoxide (DMSO) (OVX-genistein) or DMSO only (OVX-vehicle). The chosen dose of genistein was designed to represent a human diet high in soy and was scaled to a rat equivalent.²¹ Rats were studied in two separate cohorts with 18 rats (6 per group) in cohort 1 and 24 rats (8 per group) in cohort 2. Tail tendon fascicle mechanical properties were evaluated in all rats; however, tendons from cohort 1 were used for a separate set of RNA sequencing experiments. Thus, gene expression data are reported from eight rats per group.

After group assignment, rats were caged in pairs (with each pair being members of the same experimental group), allowed access to genistein-free food (Modified AIN-93G with Corn Oil; Dyets, Inc., Bethlehem, PA, USA), allowed access to water ad libitum, and maintained on a 12-h light/12-h dark cycle. After a 2-week equilibration period, rats were treated with genistein or vehicle daily for 6 weeks. After 6 weeks, rats were euthanized by decapitation after CO₂ inhalation. This investigation was approved by the Purdue University and Midwestern University Institutional Animal Care and Use Committees and all animals were cared for in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.²²

Achilles tendon gene expression

Total RNA for gene expression analysis was isolated from the Achilles tendon as previously described (Table 1).²³ Briefly, 10 to 20 mg of frozen tendon tissue was pulverized under cryogenic conditions (OPS Diagnostics, Lebanon, NJ, USA) and lysed using a bead mill homogenizer (BR12, Omni) in TRIzol Reagent (Invitrogen). Phase separation and RNA precipitation were completed per the manufacturer's instructions (TRIzol; Invitrogen). RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific). Quality of RNA was assessed using the 260/280 and 260/230 ratios. Reverse transcription (iScript; BioRad, Hercules, CA, USA) was completed to produce complementary DNA from 100 ng of RNA. Absolute quantification of mRNA target transcripts was completed using digital droplet polymerase chain reaction (ddPCR; BioRad) and reported as positive counts per 20 μL reaction.²³ Input cDNA for all genes was 1.11 ng, excluding Col1a1, Col3a1, Tnmd, and Dcn, which were 0.55 ng.

Table 1.

Digital Droplet Polymerase Chain Reaction Probes

Gene symbol	Gene name	Entrez gene
Casp3	Caspase-3	25402
Casp8	Caspase-8	64044
Ccnd1	G1/S-specific cyclin-D1	58919
Col1a1	Collagen alpha-1(I) chain	29393
Col3a1	Collagen alpha-1(III) chain	84032
Ctnnb1	Catenin beta-1	84353
Dcn	Decorin	29139
Dkk1	Dickkopf-like protein 1	22943
Esr1	Estrogen receptor α	24890
Esr2	Estrogen receptor β	25149
Mmp1a	Matrix metallopeptidase 1a precursor	300339
Mmp2	Matrix metallopeptidase 2	81686
Mmp9	Matrix metalloproteinase 9	81687
Pcna	Proliferating cell nuclear antigen	25737
Scx	Scleraxis	680712
Tgfbr3	Transforming growth factor beta receptor type 3 (betaglycan)	29610
Timp1	Tissue inhibitor of matrix metalloproteinase 1	116510
Tnmd	Tenomodulin	64104
Wnt16	Protein Wnt16 precursor	500047

Tail tendon fascicle mechanical properties

Tail fascicles (∼30 mm in length) were harvested and their mechanical properties were assessed using a Deben mini tensile tester 200 N stage (Deben, UK, Ltd.), as previously described.²⁴

Histology

Immersion-fixed (Histo-choice Fixative D, Amresco) longitudinal sections of the Achilles tendon were dehydrated through graded methanol, cleared with toluene, and embedded in paraffin (Formula R; Leica Microsystems Surgipath, Buffalo Grove, IL, USA).²⁴ Sections (4 μm) were dewaxed and rehydrated through graded ethanol and stained with hematoxylin and eosin (H&E) to determine cell density.²⁴ Total cells were counted in three representative regions of equal size and summed. Cell counting was completed independently by two investigators.

Statistical analysis

Our statistical analyses on contrasts between the intact-vehicle, OVX-vehicle, and OVX-genistein groups proceed by two classes of regression models. Normal linear regression models were fit for the tendon gene expressions data, and mixed-effects regression models were fit for the tail fascicle mechanical properties and body weight data. The independent variables that were considered for all of the regression models were indicators for the two OVX groups, with the intact group serving as the baseline. Further details on the specific regression analyses that were performed for these three types of dependent variables are provided as Supplementary Data. Our chosen significance level was α = 0.05 throughout, and we accounted for the multiple comparisons among the three groups by means of separate Bonferroni adjustments.

Results

Body mass increased with time in all groups, but rats in the OVX groups were heavier than intact rats (P < .05, Fig. 1). Tendon tail fascicle stiffness was not altered by OVX or genistein treatment (P > .05, Table 2). Deformation (P = .048), maximum stress (P = .022), and yield stress (P = .052) tended to be lower in OVX-vehicle rats when compared to intact-vehicle, but failed to reach statistical significance after correction for multiple comparisons (adjusted α = 0.017, Table 2). In contrast, energy density at yield, modulus, maximum stress, and yield stress were greater in OVX-genistein rats when compared to OVX-vehicle (P < .05, Table 2).

FIG. 1.

Rat body mass (g) over the course of the treatment protocol (n = 14). Rats were weighed weekly. *P < .05 main effect for time, all groups. ^#P < .05, intact-vehicle lower than OVX-vehicle and OVX-genistein at all time points. Data presented as mean and standard error. OVX, ovariectomy.

Table 2.

Tail Tendon Fascicle Mechanical Properties

	Intact-vehicle	OVX-vehicle	OVX-genistein
Deformation (mm)	2.62 ± 0.59	2.32 ± 0.50	2.37 ± 0.46
Energy density at yield (MJ/m3)	1.56 ± 0.63	1.36 ± 0.46	1.72 ± 0.36*
Stiffness (N/mm)	1.33 ± 0.64	1.38 ± 0.61	1.49 ± 0.66
Young's modulus (MPa)	1169 ± 285	1074 ± 277	1244 ± 240*
Maximum stress (MPa)	60.5 ± 17.7	52.1 ± 12.9	62.1 ± 12.3*
Yield stress (MPa)	49.8 ± 16.2	43.2 ± 13.7	53.9 ± 9.8*
Strain (%)	0.061 ± 0.009	0.058 ± 0.011	0.062 ± 0.005

Data are presented as means ± SE; n = 14 rats per group.

^*P < .05 versus OVX-vehicle.

OVX, ovariectomy.

Table 3 provides list of the genes evaluated, ranked by total counts in the Achilles tendon of intact rats, providing an indication of the relative abundance of each gene transcript evaluated. Dcn and Col1a1 were the genes demonstrating the greatest expression levels. Neither Col1a1 (intact-vehicle: 85,045 ± 20,981 counts per 20 μL; OVX-vehicle: 95,225 ± 7549; and OVX-genistein: 71,380 ± 13,846) nor Col3a1 (intact-vehicle: 10,568 ± 1007; OVX-vehicle: 10,243 ± 1112; and OVX-genistein: 9070 ± 810) expression was affected by OVX or genistein treatment (Fig. 2). Decorin was not influenced by OVX (intact-vehicle: 96,600 ± 6272 and OVX-vehicle: 80,625 ± 9301), but was 36% greater in genistein-treated rats (OVX-genistein: 115,800 ± 4222) when compared to OVX-vehicle (P < .05, Fig. 2). In contrast, Tgfbr3 (betaglycan) was not influenced by OVX or genistein (intact-vehicle: 1889 ± 403; OVX-vehicle: 1432 ± 198; and OVX-genistein: 1521 ± 225).

Table 3.

Genes Ranked by Counts in Intact-Vehicle Rats

Gene	Intact-vehicle
Dcn	96,600 ± 6273
Col1a1	85,045 ± 20,982
Col3a1	10,568 ± 1007
Tgfbr3	1889 ± 403
Mmp2	1756 ± 457
Tnmd	1611 ± 177
Timp1	1065 ± 81
Ctnnb1	802 ± 195
Pcna	787 ± 57
Scx	631 ± 79
Ccnd1	305 ± 77
Casp3	195 ± 33
Esr1	154 ± 29
Casp8	82 ± 12
Wnt16	17 ± 3
Mmp9	13 ± 3
Dkk1	9 ± 3
Mmp1a	7 ± 1

All counts are adjusted to per 1.11 ng of RNA.

FIG. 2.

ddPCR counts for collagen 1 (Col1a1), collagen 3 (Col3a1), decorin (Dcn), and transforming growth factor beta receptor type 3 or betaglycan (Tgfbr3). *P < .05 versus OVX-vehicle. Data presented as mean and standard error for transcript counts per 20 μL reaction. ddPCR, digital droplet polymerase chain reaction.

Gene expression of the ECM enzymes Mmp1a (intact-vehicle: 7 ± 1; OVX-vehicle: 10 ± 3; and OVX-genistein: 12 ± 2), Mmp2 (intact-vehicle: 1756 ± 457; OVX-vehicle: 2369 ± 370; and OVX-genistein: 1749 ± 326), Mmp9 (intact-vehicle: 13 ± 3; OVX-vehicle: 12 ± 3; and OVX-genistein: 15 ± 2), and Timp1 (intact-vehicle: 1065 ± 81; OVX-vehicle: 939 ± 95; and OVX-genistein: 1318 ± 82) was not influenced by OVX or genistein treatment (P > .05, Fig. 3). In contrast, Tnmd expression was 2.4-fold greater (P < .05, Fig. 4) in OVX-vehicle (3943 ± 442) when compared to intact-vehicle (1611 ± 177). Tnmd expression was 45% lower in OVX-genistein (2498 ± 115) rats when compared to OVX-vehicle, but still higher than intact-vehicle (P < .05, Fig. 4). Furthermore, Pcna expression was 33% lower in OVX-vehicle (563 ± 20) in comparison to intact-vehicle (787 ± 57, P < .05), whereas the reduction was prevented by genistein treatment (Fig. 4, 734 ± 48). Scx expression was not altered by OVX or genistein treatment (P > .05, Fig. 4, intact-vehicle: 631 ± 79; OVX-vehicle: 961 ± 85; and OVX-genistein: 1001 ± 145).

FIG. 3.

ddPCR counts for matrix metalloproteinase 1, 2, and 9 (Mmp) and tissue inhibitor of MMP 1 (Timp1), *P < .05 versus OVX-vehicle. Data presented as mean and standard error for transcript counts per 20 μL reaction. Due to the low counts of Mmp1a and Mmp9, data are also shown to a smaller scale in upper right of figure.

FIG. 4.

ddPCR counts for tenomodulin (Tnmd), scleraxis (Scx), and proliferating cell nuclear antigen (Pcna). *P < .05 versus OVX-vehicle and OVX-genistein. ^#P < .05 versus OVX-vehicle. Data presented as mean and standard error for transcript counts per 20 μL reaction.

The apoptotic genes Casp3 (intact-vehicle: 195 ± 33; OVX-vehicle: 244 ± 11; and OVX-genistein: 221 ± 21) and Casp8 (intact-vehicle: 82 ± 12; OVX-vehicle: 91 ± 13; OVX-genistein: 100 ± 10) were not influenced by OVX or genistein (P > .05, Fig. 5). Esr1 expression was greater (P < .05, Fig. 5) in OVX-vehicle (307 ± 47) compared to intact-vehicle (154 ± 29). Esr1 expression also tended to be higher (twofold) in OVX-genistein rats (271 ± 31, P = .023) when compared to intact-vehicle, but this effect did not reach statistical significance after correcting for the multiple comparisons. Esr2 transcripts were not detected using the predesigned Biorad probe. Wnt16 (intact-vehicle: 17 ± 3; OVX-vehicle: 34 ± 6; and OVX-genistein: 26 ± 5), Ccnd1 (intact-vehicle: 305 ± 77; OVX-vehicle: 385 ± 47; and OVX-genistein: 420 ± 88), Ctnnb1 (intact-vehicle: 802 ± 195; OVX-vehicle: 966 ± 134; and OVX-genistein: 875 ± 160), and Dkk1 (intact-vehicle: 9 ± 3; OVX-vehicle: 10 ± 2; and OVX-genistein: 9 ± 3) expression were not altered by OVX or genistein treatments (P > .05, Fig. 6). Cell counts from the H&E stains were not influenced by OVX or genistein (P > .05). Although an extensive histological evaluation of morphology was not conducted, no gross difference in morphology across groups was noted (Fig. 7).

FIG. 5.

ddPCR counts for caspase 3 and 9 (Casp) and estrogen receptor 1 (Esr1). *P < .05 versus OVX-vehicle. Data presented as mean and standard error for transcript counts per 20 μL reaction.

FIG. 6.

ddPCR counts for Wnt family member 16 (Wnt16), cyclin D1 (Ccnd1), catenin beta 1 (Ctnnb1), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk1). Data presented as mean and standard error for transcript counts per 20 μL reaction. Due to the low counts of Wnt16 and Dkk1, data are also shown to a smaller scale in upper right of figure.

FIG. 7.

Achilles tendon cell density as determined from Achilles tendons sections (4 μm) stained with H&E. Data presented as mean ± standard error. Representative histology panels are provided for each group. H&E, hematoxylin and eosin.

Discussion

In our previous work, we demonstrated that loss of estrogen due to OVX leads to a large decline in rat Achilles tendon collagen, an effect that is prevented by administration of genistein.⁹ In this investigation, we follow up on these findings by determining the impact of OVX and genistein treatment on tendon functional properties, while also exploring the effects of OVX and genistein on tendon ECM regulators. In line with our Achilles tendon collagen findings,⁹ tendon fascicle failure stress tended to be lower after OVX and genistein treatment led to higher failure stress. Tendon fascicle modulus and energy density were also modestly higher in OVX-genistein-treated rats. Surprisingly, even with the large loss of Achilles tendon collagen noted in our previous work,⁹ we did not observe an effect of OVX or genistein on Achilles tendon Col1a1 or Col3a1 expression. Furthermore, key genes associated with ECM degradation were not altered by OVX or genistein treatment. We did, however, observe a modest increase in Dcn expression, a proteoglycan involved with regulating collagen fibril formation²⁵ with genistein treatment. Although no effect of OVX or genistein on the expression of Wnt pathway-associated genes was noted, we did observe significant effects of OVX and genistein on Tnmd and Pcna expression, which are key modulators of cellular proliferation.²⁶ Based on our gene data, we hypothesize that the effects of OVX and genistein on tendon collagen may be driven more by changes in cellular proliferation genes rather than direct effects on expression of ECM-related genes. Future studies with more specific measures of cellular proliferation and enzyme activity are needed to further investigate this hypothesis.

Collagen is the primary protein constituent of tendon and correlates strongly with tissue mechanical properties.¹¹ Therefore, it seemed likely that tendon fascicle mechanical properties would be impaired in OVX rats, given our previous report demonstrating a large loss of tendon collagen with OVX.⁹ Consistent with limited previous studies,¹² tendon fascicle failure stress tended to be lower after OVX. Genistein treatment also resulted in higher modulus and energy density when compared to untreated OVX rats. However, we did not observe effects on several other critical functional variables such as stiffness. Changes in tendon stiffness can alter musculoskeletal performance during functional activities,²⁷ but our findings suggest that loss of estrogen may not alter tendon function under submaximal loading conditions. Importantly, genistein administration, at amounts easily obtained in the diet, was effective at preserving tendon fascicle failure stress to similar levels as those of intact rats. Our findings provide preclinical evidence that genistein, at doses that can be obtained from dietary consumption, may be useful for minimizing the impact of estrogen loss on tendon functional properties. The conclusions of our mechanical findings should be considered with some caution as we are comparing our mechanical property assessment from tail tendon fascicles to our previous collagen content and crosslink findings in the Achilles tendon.⁹

A second objective of this investigation was to further develop our understanding of the mechanisms mediating the effects of estrogen loss and genistein on tendon. Given the large decline in total tendon collagen noted previously,⁹ we examined the impact of OVX and genistein on Col1a1 and Col3a1 expression as well as expression of the proteoglycans decorin and betaglycan (Tgfbr3). Surprisingly, we did not observe an effect of OVX or genistein on the expression of collagen genes. This finding suggests that changes in collagen content due to OVX or genistein may be mediated by post-translational modifications of collagen synthesis or breakdown, rather than alteration of gene transcription.

Although betaglycan expression was not altered by either OVX or genistein, Dcn expression was higher in OVX-genistein-treated rats relative to OVX-vehicle. Dcn is the most abundant proteogylcan in tendon and a highly expressed transcript (Table 3), with roles modulating fibrillogenesis and mechanical properties.^25,28–30 Consistent with our findings, steroids, such as dexamethasone, have been shown to stimulate decorin expression in fibroblasts without altering betaglycan expression.³¹ While we are not aware of any previous investigation in tendon demonstrating an ability of genistein to modify decorin transcription or other proteoglycans, genistein has been shown to inhibit versican expression, but not decorin and biglycan, in arterial smooth muscle cells.³² Regardless, genistein had only modest effects of proteoglycan expression in our model, and further work is needed to determine if this effect contributes to the noted improvements in tendon function and collagen content.

In addition to collagen and proteoglycans, a large family of matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) regulate degradation of ECM components, including collagen. Previous work by Pereira et al.³³ reported that MMP-2 enzyme activity was greater in the rat Achilles tendon after 3 months of OVX when compared to intact rats. Furthermore, in non-tendon models, genistein has been shown to inhibit induction of MMPs, which degrade collagen and other ECM components.³⁴ The combination of these studies yields the inference that estrogen and genistein may modulate expression of some MMPs. Surprisingly, we did not observe any change in the expression of several common genes encoding MMP and TIMP enzymes. Comparisons to this study are difficult due to differences in tissue type, rat age, and strain. Furthermore, we only evaluated tendon gene expression, limiting our comparisons to Pereira et al.³³ As with our collagen interpretation, it is possible that the effects of OVX on MMPs could be post-translational; thus, further studies are needed to carefully examine genistein's impact on ECM enzyme activity.

We also determined the impact of OVX and genistein treatment on cell proliferation markers and expression of genes related to the β-catenin/Wnt signaling pathway. Tnmd and Scx are well-established tendon-specific genes that have several roles in the modulation of fibrillogenesis and cell proliferation.^35,36 In addition, Pcna is a common marker of cell proliferation. Although we did not observe a significant effect of OVX or genistein on total cell counts or Scx expression, Tnmd expression was greater in OVX-vehicle rats when compared to intact-vehicle. Genistein treatment resulted in lower Tnmd expression relative to OVX-vehicle, but Tnmd expression under OVX-genistein was still greater than intact-vehicle. Pnca expression was lower in OVX-vehicle rats when compared to intact-vehicle, but its expression was similar when comparing intact-vehicle- and OVX-genistein-treated rats. The β-catenin/Wnt signaling pathway has not been extensively examined in tendon, but has been shown to modulate Tnmd and Scx in tendon-derived cells.^37,38 Activation of Wnt/β-catenin signaling in tendon-derived cells lead to suppressed expression of Scx and Tnmd.³⁷ However, we found no effect of estrogen loss or genistein treatment on expression of Wnt/β-catenin-related genes. Furthermore, in our model, Tnmd and Scx responded differently to OVX and genistein, suggesting that loss of estrogen may not modulate Wnt/β-catenin signaling in tendon.

Interestingly, we noted an increase in Esr1 expression after OVX, an effect that was not normalized by genistein treatment. Work in other tissues observed an increase in Esr1 expression after OVX, although tissue specific.³⁹ Furthermore, using validated Esr2 probes (Biorad), we were not able to detect expression of Esr2 transcripts in the Achilles. We have also noted no expression of Esr2 in the Achilles tendon using RNA sequencing (unpublished observation). In contrast, others have reported the presence of Esr2 protein in tendon-derived cells with greater expression after OVX.⁴⁰ Genistein is an estrogen receptor agonist and has a much greater affinity for ER-beta (Esr2).⁴¹ If ER-beta is indeed not present in rat tendon tissue, genistein may modulate tendon collagen and ECM gene expression through non-estrogenic pathways.

Of note, ddPCR technology allows for absolute quantification of mRNA transcripts with a detection limit of a single transcript. Although the relationship between overall transcript expression in a tissue and the importance of a gene to tissue cellular function is not clear, our ddPCR data (Table 3) do provide an interesting observation regarding tenocyte “commitment” to generating transcripts for a specific gene. As might be expected, the number of collagen and decorin transcripts was considerably higher than the other genes evaluated. Interestingly, in intact rats, genes such as Tnmd and Scx were expressed at a level of ∼50–250-fold less than Col1a1. Expression of Mmp9, an important modulator of ECM breakdown, which has been examined in several tendon-related studies, was surprisingly very low.

In conclusion, we demonstrate that OVX led to a decline in stress at tendon fascicle failure, but other important functional variables (e.g., stiffness) were not impacted. We can also conclude that genistein can reverse the effects of OVX on tendon fascicle failure stress. Even though we previously observed a large reduction in tendon collagen content after OVX,⁹ our current findings infer that these changes may, in part, be modulation of Tnmd and Pcna, rather than collagen genes or expression of ECM-degrading proteins. Furthermore, our findings suggest that genistein, although shown to have an affinity for estrogen receptors, does not completely reverse the effects of estrogen loss on the expression of several genes in the rat Achilles tendon. Future studies should examine the impact of combined estrogen and genistein supplementation and include protein measures and evaluation of estrogen receptor activation. In addition, future work should also consider biological mechanisms that occur independent of estrogen receptor signaling, as they may reveal alternative targets for genistein. Further work would help to evaluate and justify the inclusion of genistein supplementation or dietary modifications to include soy isoflavones, which could potentially aid in reducing use of pain relieving medication in postmenopausal women suffering from painful tendinopathies and ultimately improve quality of living.

Disclaimer

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This publication was funded, in part, by NIH R15 AT00860501A1 to C.C.C., Purdue University Research Initiative Funds to C.C.C, and with support from the Indiana Clinical and Translational Sciences Institute funded, in part, by award number UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. S.H.P was supported by NIH F31-AR073647.

Supplementary Material

Supplementary Data