In vitro modulatory effect of dehydroepiandrosterone sulfate on apoptosis and expression of apoptosis‐related genes in patients with systemic lupus erythematosus

Abstract

Objectives

Systemic lupus erythematosus‌ (SLE) is an autoimmune disease characterized by hyperactive B cells that produce various autoantibodies. Sex hormones have been documented to influence the development of SLE, in which women with SLE have low plasma level of dehydroepiandrosterone sulfate (DHEAS). A strong conclusion about the effect of DHEAS on apoptosis in SLE patients has not been provided. The aim of this study was to assess apoptotic effects of DHEAS on peripheral blood lymphocytes (PBLs) from SLE patients.

Methods

Twenty SLE patients and 20 age‐ and sex‐matched healthy controls were included into this study. Concentration of DHEAS was measured using enzyme‐linked immunosorbent assay in serum from all participants. Freshly isolated PBLs from each individual were treated with 7.5‐µmol of DHEAS for 24 hr in cell culture medium to assess the effect of DHEAS on apoptosis using fluorescein isothiocyante‐conjugated annexin V and propidium iodide. The messenger RNA (mRNA) expression level of apoptosis‐related genes (Fas, Fas‐L, Bcl‐2, and Bax) in PBLs was measured using real‐time PCR before and after treating with DHEAS.

Results

Level of DHEAS was low in SLE patients compared with healthy controls (p < 0.05). After treating with DHEAS, the percentage of apoptotic cells in SLE patients was decreased in comparison with healthy controls. DHEAS treatment increased the mRNA expression level of Bcl‐2 in PBLs from SLE patients.

Conclusions

DHEAS reduced the apoptosis rate in PBLs from SLE patients and may decrease the load of autoantigens. Therefore, DHEAS might be considered as a therapeutic tool in SLE patients.

Dehydroepiandrosterone sulfate (DHEAS) reduced the apoptosis rate in peripheral blood lymphocytes from systemic lupus erythematosus (SLE) patients, and may decrease the load of autoantigens. Therefore, DHEAS might be considered as a therapeutic tool in SLE patients.

1. INTRODUCTION

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder that has a significantly female predominance with the female to male ration of 9:1 (Petri, 2008). Immune cells in SLE patients become autoreactive to their own antigens. The main reason for autoreactivity of immune cells results from stimulation of cells by intracellular components because of abnormal apoptosis. It has been established that autoantibodies, cytokines, and sex hormones are involved in the impaired apoptosis procedure (Licht, Dieker, Jacobs, Tax, & Berden, 2004; Marks & Tullus, 2012; Mor et al., 2003; Ren et al., 2003). On the other side, apoptosis, by clearing the damaged cell debris prevents entry of released intracellular material that have inflammatory effects on tissues (Elmore, 2007). Apoptotic cells, under normal circumstances, are efficiently uptaken by macrophages without stimulating the inflammatory or any other immune response (Charles, 2003). Investigations suggest that defective immune regulatory mechanisms like diminished clearance of apoptotic cells and immune complexes, contribute to the development of SLE (Navratil, Liu, & Ahearn, 2006). The causes for explaining such impairments in SLE are environmental, epigenetic, and genetic factors (Ebrahymian et al., 2018; Fathollahi, Gabalou, & Aslani, 2018; Foma, Aslani, Karami, Jamshidi, & Mahmoudi, 2017; Mahmoudi et al., 2017; Salmaninejad et al., 2017).

Dehydroepiandrosterone (DHEA) is a pregnenolone‐derived C19 steroid, which is synthesized mainly in the zona reticularis of the human adrenal gland (Endoh, Kristiansen, Casson, Buster, & Hornsby, 1996). DHEA, in response to stimulation by the endogenous hormone adrenocorticotrophin, is secreted from the adrenal cortex alongside its sulfated precursor, namely dehydroepiandrosterone sulfate (DHEAS). These steroids, together, are the most abundant in human circulation, which reach peak concentrations during the third decade of life (Migeon, Keller, Lawrence & Shepard, 1957; Orentreich, Brind, Vogelman, Andres, & Baldwin, 1992). DHEAS levels is higher than DHEA in the body, which is attributable to its slower rate of metabolic clearance as well as longer half‐life (Longcope, 1996). Studies demonstrate that serum DHEA or DHEAS is significantly lower in SLE patients compared with controls (Suzuki, Suzuki, Engleman, Mizushima, & Sakane, 1995). Furthermore, it has been shown that administration of DHEA to the patients with SLE has therapeutic potential (D. M. Chang, Lan, Lin, & Luo, 2002). DHEA's beneficial effects may be exerted through increasing serum androgen levels as well as increasing serum estradiol concentrations (Barry, McGuire, & Van Vollenhoven, 1998).

The absolute number of lymphocytes in males and females is almost similar; however, the percentage of T cells is lower in males (Bouman, Schipper, Heineman, & Faas, 2004). This is somehow associated with the effect of testosterone on the apoptosis of T cells (McMurray, Suwannaroj, Ndebele, & Jenkins, 2001). Moreover, it has been established that estrogens inhibit autoreactive B cell apoptosis and, therefore, increase their survival in mice, which could be related to a high prevalence of autoimmune diseases in females (Grimaldi, Cleary, Dagtas, Moussai, & Diamond, 2002). The inhibition of apoptosis of peripheral blood lymphocytes (PBLs) of SLE patients by DHEA has been documented (Takahashi, Nakajima, & Sekihara, 2004). On the other side, studies show that DHEA could enhance the expression of Fas and Fas‐L and induces thymocyte apoptosis in patients with SLE (Liang, Yao, Yang, & Hou, 2004). Given the controversial finding about the role of DHEAS on the apoptosis of SLE PBLs, this study was performed to unravel the mechanism by which DHEAS may affect the PBLs from patients with SLE.

2. MATERIALS AND METHODS

2.1. Study subjects

Twenty SLE patients and 20 sex‐ and age‐matched healthy individuals were recruited into the study. All patients fulfilled the American College of Rheumatology classification criteria for SLE (Hochberg, 1997) and were diagnosed as having SLE with the SLE disease activity index score > 10, indicating active disease form (Bombardier et al., 1992). Patients had received no immunomodulatory treatment at least 3 months before entering the study. No cytotoxic drug was administered to patients. Female participants were in the middle of their menstrual cycles and had not used any contraceptive drugs. Pregnant women were excluded from the study. Healthy individuals included 20 subjects with no other autoimmune diseases, cancer, and recent infection. This study was approved by the Ethical Committee of the Mashhad University of Medical Sciences. All participants completed the informed written consents before entering into the research.

2.2. Blood collection, lymphocyte isolation, and cell culture

In this study 20 ml of venous peripheral blood was obtained from each subject. Out of which, 18 ml was transferred into sterile tubes containing EDTA 10% for lymphocyte isolation procedures and 2 ml was used for serum isolation. To isolate lymphocytes, a mixture of 1:1 proportion of whole blood phosphate buffered saline (PBS) was implemented. Afterwards, 6 ml of the diluted blood was poured slowly over 3 ml of Ficoll (Cedarlane Lab; Hornby, Ontario, Canada) solution and then centrifuged for 20 min at 1200 rpm and subsequently for 20 min at 1550 rpm in 25°C. The cells were washed twice in PBS and then the cell pellets resuspended in 1 ml of PBS. The resuspended peripheral blood mononuclear cells (PBMCs) were transferred into 25 ml polyester flasks containing 5 ml RPMI‐1640 (Biosera, East Sussex, UK) with 10% FCS (Biosera). Isolated PBMCs were incubated at 37°C in the presence of 5% CO₂ for 2 hr. Afterwards, nonadherent lymphocytes were collected, and 1 × 10⁶ cells were cultured in complete 1640 medium containing 10% FCS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated at 37°C with 5% CO₂ for 24 hr in the presence or absence of 7.5 µM of DHEAS (Sigma, St. Louis, MO).

2.3. Apoptosis detection by flow cytometry

For measuring the apoptosis, 100 µl of cultured lymphocytes from treated and untreated suspensions were washed in calcium buffer and then poured off in flow cytometry tubes for assessment of apoptosis using Phosphatidyl Serine Detection Kit (IQ Products, Groningen, The Netherlands). After that, 5 µl of fluorescein isothiocyante (FITC)‐conjugated annexin V (annexin V‐FITC) was added to the cell suspension of each tubes, gently mixed, and incubated for 20 min at 4°C in a dark chamber. Then, cells were washed with calcium buffer and 5 µl of propidium iodide (PI) was added into each tube and kept for 10 min at 4°C in a dark chamber. After staining, cells were washed twice and resuspended in PBS. Then, 1 × 10⁴ cells/sample were gated and evaluated by green fluorescence (FL1‐H) filter using untreated cells as a control in a FACSCalibur^TM System (Becton Dickinson). The CellQuest Software (Becton Dickinson) was used for data analysis.

2.4. DHEAS levels in serum

After serum isolation, the serum DHEAS concentration was measured through enzyme linked immunosorbent assay using a commercial kit (Biosource Europe SA, Nivelles, Belgium).

2.5. RNA extraction from lymphocytes

Total cellular RNA was extracted using High Pure RNA Isolation Kit (Roche, Mannheim, Germany) according to manufacturer's manuals. Yield and purity of RNA were determined using a NanoDrop spectrophotometer at 260/280 nm (NanoDrop ND‐2000C Spectrophotometer; Thermo Fisher Scientific).

2.6. Reverse transcription and complementary DNA synthesis

First‐strand complementary DNA (cDNA) was synthesized from the RNA of cells using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Briefly, first 4 µl of isolated RNA (30 µg) through previous procedure was mixed with 1 µl of random hexamer primer and 7 µl RNASe‐free H₂O and then incubated at 65°C for 5 min. Afterwards, micro tubes were chilled on ice, and a mixture of reaction buffer 4 µl, RNase inhibitor 1 µl, dNTP mix 2 µl, and reverse transcriptase 1 µl were added to each sample. Samples were immediately incubated at 25°C for 5 min followed by 42°C for 60 min; the reaction was finally terminated by heating at 70°C for 5 min. Reverse transcription was performed with the final volume of 20 µl pretube.

2.7. Real‐time quantitative polymerase chain reaction

Quantitative analysis of messenger RNA (mRNA) expression levels of Fas, Fas‐L, Bcl‐2, and Bax before and after treatment was carried out by Real Time PCR using the TaqMan Gene Expression Assays containing the FAM dye‐labeled probes (TaqMan Pre‐designed Gene Expression Products; Applied Biosystems, Foster City, CA) and StepOne Plus Real‐Time PCR System (Applied Biosystems). Sequence of primers and probes used in this study are shown in Table 1. Each reaction mixture contained a total volume of 20 μl (master mix 10 μl, cDNA 2 μl [5 ng/ml], assay mix 1 μl, and H₂O 7 μl). The quantitative RT‐PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s, and 60°C for 1 min. A widely used approach to represent relative gene expression, the comparative C _t method, was exploited to evaluate expression as previously described by Schmittgen and Livak (Schmittgen & Livak, 2008). Relative amounts of target mRNAs in test sample were calculated and normalized to the corresponding β‐Actin mRNA transcript level as a housekeeping gene. Then relative expression for each sample was calculated using the following equation: relative mRNA expression = $\mathrm{\text{‐}}\left(2^{-\mathrm{\Delta }{\mathit{C}}_{\mathrm{t}}}\right)\times {10}^3$.

Table 1.

Primers and probes used for gene expression analysis by real‐time PCR

Target genes	Accession number	Sequence
G6PDH	NM_000402	Forward	5′‐AgCCgggCATgTTCTTCAAC‐3′
		Reverse	5′‐AgggAgCTTCACgTTCTTgTATC‐3′
		Probe	5′‐CggAgCTggACCTgACCTACggCA‐3′
Fas‐L	NM_000639	Forward	5′‐CACACAgCATCATCTTTggAgAAg‐3′
		Reverse	5′‐TggACCTTgAgTTggACTTgC‐3′
		Probe	5′‐AAATAggCCACCCCAgTCCACCCC‐3′
Fas	NM_000043	Forward	5′‐gATgAAggACATggCTTAgAAgTg‐3′
		Reverse	5′‐ggTCACAgTgTTCACATACAgTAg‐3′
		Probe	5′‐ACTgCACCCggACCCAgAATACCA‐3′
Bcl‐2	NM_138761	Forward	5′‐gTgTgTggAgAgCgTCAACC‐3′
		Reverse	5′‐TCCCAgCCTCCgTTATCCTg‐3′
		Probe	5′‐CgCCCCTggTggACAACATCgCC‐3′
Bax	NM_000633	Forward	5′‐TTgTCgCCCTTTTCTACTTTgC‐3′
		Reverse	5′‐TCCAATgTCCAgCCCATgATg‐3′
		Probe	5′‐AAACTggTgCTCAAggCCCTgTgC‐3′

Note. Bcl‐2: B‐cell lymphoma 2; Fas‐L: Fas ligand; G6PDH: glucose‐6‐phosphate dehydrogenase; PCR: polymerase chain reaction.

2.8. Statistical analysis

Data analysis was fulfilled via SPSS software (version 21; SPSS, Chicago, IL). The Mann–Whitney U test was performed for comparing the mean percentage of apoptosis, mRNA expression level, and DHEAS level between patients and controls. Also, Wilcoxon test was performed to compare the apoptosis rate and mRNA expression level between the untreated and treated cells within each group. The GraphPad Prism Version 5.00 for Windows (GraphPad Software, La Jolla CA; www.graphpad.com) was applied to illustrate data through graphs. All results are expressed as mean ± standard error of mean with statistical significance set at 5%.

3. RESULTS

Clinical specification of patients and healthy controls are listed in Table 2. SLE patients with the mean age of 33.05 ± 8.3 were age‐matched with healthy individuals with mean age of 32.8 ± 8.27. Both groups were consisted of three males and 17 females. The DHEAS concentration in the serum of patients was 0.7 ± ‌0.7 µl/ml, which significantly (p = 0.001) differed from that of healthy controls with 1.85 ± 0.88 µl/ml. Complete blood count (CBC) indices were all in the normal rage for SLE patients. Erythrocyte sedimentation rate, anti‐dsDNA, C3, and C4 levels is SLE patients were 26.82 ± 11.3 mm/hrs, 294.1 ± 275.1 IU/ml, 103.3 ± 43.39 µg/ml, 20.65 ± 12.12 µg/ml, respectively. Ten SLE patients were positive for C‐reactive protein (CRP), four cases for rheumatoid factor (RF), and 17 cases for antinuclear antibody (ANA). Four SLE patients had familial history of SLE in their family members. There were five pregnant SLE cases among the study population. Four cases of female SLE patients had at least one times of abortion experience. Finally, proteinuria was observed in four SLE patients. Specification of patients are listed in Table 2 with more details.

Table 2.

Clinical specification of systemic lupus erythematosus patients and healthy subjects

Characteristics	Patients (N (%)/mean ± SD)	Healthy subjects (N (%)/mean ± SD)
Age	33.05 ± 8.3	32.8 ± 8.27
Sex
Male	3 (15)	3 (15)
Female	17 (85)	17 (85)
DHEAS (µl/ml)	0.7 ±  0.7	1.85 ± 0.88
RBC	5 ± 0.52 × 106	—
WBC	6.05 ± 2.22 × 103	—
Platelet count	185.8 ± 80.4 × 103	—
ESR	26.82 ± 11.3	—
Anti‐dsDNA (IU/ml)	294.1 ± 275.1	—
C3 (µg/ml)	103.3 ± 43.39	—
C4 (µg/ml)	20.65 ± 12.12	—
CRP
Positive	10	—
Negative	10	—
RF
Positive	4	—
Negative	16	—
ANA
Yes	17	—
No	3	—
Familial history
Yes	4	—
No	16	—
Pregnant women
Yes	5	—
No	12	—
Abortion
Yes	4	—
No	13	—
Proteinuria
Yes	4	—
No	16	—

Note. ANA: antinuclear antibody; CRP: C‐reactive protein; DHEAS: dehydroepiandrosterone sulfate; ESR: erythrocyte sedimentation rate; RF: rheumatoid factor; SD: standard deviation.

3.1. Early and late apoptosis rate in DHEAS‐treated and untreated PBLs from patients with SLE

The percentage of the early apoptotic cells (annexin V‐FITC⁺/PI⁻ cells; Figure 1) for untreated PBLs in SLE patients (14.0 ± 0.8%) was significantly (p = 0.002) higher than healthy controls (9.2 ± 1.2%; Table 3 and Figure 2a). Late apoptotic cells (annexin V‐FITC⁺/PI⁺ cells) percentages in SLE patients and controls were 11.1 ± 1.3% and 8.4 ± 1.3%, respectively. There was a significant difference of the late apoptosis rate between patients and healthy control group (p = 0.023; Table 3 and Figure 2b).

Figure 1.

Representative analysis of apoptosis by two‐parameter annexin‐V FITC/PI flow cytometry of PBLs. The proportion of FITC+/PI− cells corresponds to early apoptosis, and that of FITC+/PI+ cells corresponds to late apoptosis or secondary necrosis. (a) Untreated PBLs. (b) Incubation with DHEAS (7.5 μM). DHEAS: dehydroepiandrosterone sulfate; FITC/PI: fluorescein isothiocyante/propidium iodide; PBL: peripheral blood lymphocyte

Table 3.

Apoptosis rate of treated and untreated PBLs from systemic lupus erythematosus patients and healthy subjects

	Early apoptosis			Late apoptosis
	SLE	HS	p Value	SLE	HS	p Value
Untreated (%)	14.0 ± 0.8	9.2 ± 1.2	0.002	11.1 ± 1.3	8.4 ± 1.3	0.023
DHEAS‐treated (%)	12.2 ± 0.73	9.1 ± 1.1	0.013	10.3 ± 1.2	8.8 ± 0.9	0.09
p Value	0.012	0.211	0.187	0.241

Note. DHEAS: dehydroepiandrosterone sulfate; HS: healthy subjects; SLE: systemic lupus erythematosus. Bold items demonstrate statistically significant values.

Figure 2.

Apoptosis rate comparison between different groups. (a,c) Early apoptosis. (b,d) Late apoptosis. HS: healthy subjects; SLE: systemic lupus erythematosus

3.2. Early and late apoptosis rate in DHEAS‐treated and untreated PBLs from healthy controls

The early apoptosis rate of DHEAS‐treated PBLs was 9.1 ± 1.1% in healthy subjects and that was 12.2 ± 0.73% in SLE patients. There was a significant difference in early apoptosis rate of PBLs after treating with DHEAS between patients and controls (p = 0.013; Figure 2a). The late apoptosis rate of PBLs after treating with DHEAS in patients and healthy controls were 10.3 ± 1.2% and 8.8 ± 0.9%, respectively, and the difference was insignificant (p = 0.09; Figure 2b).

3.3. Comparison of early and late apoptosis rate in DHEAS‐treated and untreated PBLs from patients with SLE and healthy controls

The early apoptosis rate of PBLs in SLE patients before and after treatment was 14.0 ± 0.8% and 12.2 ± 0.73%, respectively, which demonstrated significant (p = 0.012) reduction in apoptosis rate of PBLs after treating with DHEAS (Table 3 and Figure 2c). However, no significant alteration was observed in the late apoptosis rate of PBLs in SLE patients before and after treatment (11.1% ± 1.3% and 10.3% ± 1.2; p = 0.187; Figure 2d).

The early apoptosis rate of PBLs in healthy individuals before and after treatment did not differ significantly (9.2% ± 1.2 and 9.8 ± 0.9; p = 0.211; Table 3; Figure 2c). Although, the late apoptosis rate of PBLs in healthy subjects after treatment was 8.8 ± 180.1 and showed slight increase compared with before treatment with the rate of 8.4% ± 1.3, the difference was insignificant (p = 0.241; Figure 2d).

3.4. Gene expression of Fas, Fas‐L, Bcl‐2, and Bax in DHEAS‐treated and untreated PBLs from patients with SLE

No significant difference was observed in the mRNA expression level of Fas (fold change = 1.12; p = 0.338), Fas‐L (fold change = 0.89; p = 0.498), Bcl‐2 (fold change = 1.10; p = 0.878), and Bax (fold change = 1.17; p = 0.658) in DHEAS‐treated PBLs of healthy individuals compared with untreated PBLs of healthy individuals (Table 4, Figure 3a).

Table 4.

Fold changes of Fas, Fas‐l, Bax, and Bcl‐2 expressions in patients with systemic lupus erythematosus in comparison with healthy individuals

	Fold change
	DHEAS‐treated versus untreated		SLE versus HS
	SLE	HS	Untreated	DHEAS‐treated
Fas	1.55 (p = 0.128)	1.12 (p = 0.338)	0.89 (p = 0.612)	1.10 (p = 0.851)
Fas‐L	1.77 (p = 0.120)	0.89 (p = 0.498)	1.11 (p = 0.421)	2.27 (p = 0.027)
Bax	1.69 (p = 0.109)	1.17 (p = 0.658)	1.18 (p = 0.814)	1.87 (p = 0.074)
Bcl‐2	2.35 (p = 0.017)	1.10 (p = 0.878)	1.15 (p = 0.845)	2.14 (p = 0.044)

Note. Bcl‐2: B‐cell lymphoma 2; DHEAS: dehydroepiandrosterone sulfate; Fas‐L: Fas ligand; HS: healthy subjects; SLE: systemic lupus erythematosus. Bold items demonstrate statistically significant values.

Figure 3.

Relative expression of Fas, Fas‐L, Bax, and Bcl‐2 mRNA in four surveyed groups. Graphs (a) and (b) show expression level in DHEAS‐treated PBLs versus untreated PBLs. Graphs (c) and (d) illustrate expression level in SLE PBLs versus PBLs from healthy subjects. Bcl‐2: B‐cell lymphoma 2; DHEAS: dehydroepiandrosterone sulfate; Fas‐L: Fas ligand; HS: healthy subjects; mRNA: messenger RNA; PBL: peripheral blood lymphocyte; SLE: systemic lupus erythematosus

3.5. Gene expression of Fas, Fas‐L, Bcl‐2, and Bax in DHEAS‐treated and untreated PBLs from patients with SLE and comparison with healthy controls

Although the mRNA expression level of Fas (fold change = 1.55; p = 0.128), Fas‐L (fold change = 1.77; p = 0.12), and Bax (fold change = 1.69; p = 0.109) had no significant difference between PBLs from SLE patients before and after treatment, mRNA expression level of Bcl‐2 was upregulated in PBLs of SLE patients after treating with DHEAS in comparison to before treatment (2.35 times upregulated; p = 0.017; Table 4 and Figure 3b).

3.6. Gene expression of Fas, Fas‐L, Bcl‐2, and Bax in DHEAS‐treated and untreated PBLs from healthy controls

In comparison with healthy individuals, SLE patients expressed Fas‐L, Bcl‐2, and Bax mRNA in untreated PBLs insignificantly high (fold change = 1.11, 1.15, and 1.18; p = 0.421, 0.845, and 0.814, respectively). Furthermore, no significant difference was observed in mRNA expression level of Fas in untreated PBLs from SLE patients compared with healthy participants (fold change = 0.89; p = 0.621), despite lower expression in SLE patients (Table 4 and Figure 3c).

The mRNA expression levels of Fas‐L and Bcl‐2 were significantly upregulated in PBLs of SLE patients after being treated with DHEAS in comparison with the treated PBLs of healthy controls (2.27 and 2.14 times upregulated; p = 0.027 and 0.044, respectively). Although insignificant (Table 4 and Figure 3d), mRNA expression level of Fas and Bax showed higher expression in treated PBLs of SLE patients compared with healthy subjects (fold change = 1.10 and 1.87; p = 0.851 and 0.074, respectively).

4. DISCUSSION

Currently, there is a consensus that the strongest risk factor for development of SLE is female sex and the female‐to‐male sex ratio of 9:1 in SLE has been reported (Petri, 2002). Since SLE occurs predominantly in females, it seems that the disease development might be related to the role of sex and hormonal factors and their interactions with the immune system. Sex hormones appear to play an important roles in the type of immune response and onset of autoimmune diseases (Foo et al., 2017; Grover et al., 2016). Female hormones have strong influence on humoral immunity and inflammatory reactions, which is mediated by CD4 + Th2 lymphocytes. However, androgens as natural immunosuppressive factors, downregulate T‐ and B‐cell interactions and promote cell‐mediated cytotoxic processes through activation of CD4+ Th1 and CD8+ lymphocytes (Kissick et al., 2014). Aberrant regulation of hormonal metabolisms are common in SLE patients, as such males and females with SLE have decreased availability of androgens and increased level of estrogens (Sciascia et al., 2017; Golder and Hoi, 2017).

In addition to sex and hormones, dysfunction of apoptosis mechanisms seems to play a major role in the pathogenesis of SLE. Apoptosis of lymphocytes is increased in SLE patients and there is a defective clearance of apoptotic cells, which may lead to a pathological accumulation of abnormal cell debris. As a result, a source of available autoantigens, provide a conditions that have a potential effect on the initiation and perpetuation of autoimmune response (Tsokos et al., 2016; Truszewska et al., 2017; Yang et al., 2015). Low serum level of DHEA in SLE patients has been shown, which is in accordance with increased rate of lymphocyte apoptosis.

In our study, on the other side, significantly decreased DHEAS in serum of SLE patients was observed. Furthermore, upon treating with DHEAS, SLE PBLs decreased the apoptosis rate. These findings suggest that 7.5 µM of DHEAS is efficient in reducing the apoptosis of PBLs in SLE patients and, therefore, may provide amelioration of disease manifestation due to declining autoantigens and autoimmune responses. Moreover, the upregulation of Bcl‐2 mRNA in the PBLs of SLE patients after treating with DHEAS was observed. BCL2 gene encodes an integral outer mitochondrial membrane protein which acts as a suppressor of apoptosis of some cells, such as lymphocytes, and is classified as oncogenes (Boise et al., 1993). To conclude prematurely, DHEAS may decrease apoptosis of SLE PBLs through upregulation of Bcl‐2. However further studies may unveil the precise mechanisms by which DHEAS contributes to reduced apoptosis rate and prevents disease exacerbation. Furthermore, the mechanisms underlying the effect of DHEAS in upregulation of the Fas‐L and Bcl‐2 in PBLs from SLE patients, but not from healthy individuals, remain unclear.

Other than apoptosis, DHEA participates in modulation of inflammation and thus might be involved in the development of SLE or exacerbation of the disease course. DHEA level have been reported to have negative correlation with interleukin (IL)‐6 level (Straub et al., 2002). IL‐6 is considered as a pleiotropic cytokine and has strongly been implicated to be involved in etiopathogenesis of lupus nephritis (Iwano et al., 1993; Peterson, Robertson, & Emlen, 1996). Moreover, a bulk of investigations has established that there is an improvement in the function of immune cells upon supplementation with DHEA that is manifested as downmodulation of proinflammatory cytokines like IL‐1 and tumor necrosis factor α (D. Chang, Chu, Chen, Kuo, & Lai, 2004; Dillon, 2005). In addition, clinical trials have demonstrated that DHEA is beneficial in improving the clinical presentation of SLE patients as measured by SLE disease activity index and physician global assessment of disease activity (Durcan & Petri, 2016). These observations may suggest that DHEA might improve the disease course by modulating the cytokine profile in SLE patients.

It is concluded that DHEAS decrease apoptosis rate of SLE PBLs perhaps through upregulation of antiapoptotic molecule of Bcl‐2. Therefore, the replacement therapy of DHEAS and androgens may have beneficial effects in amelioration of disease manifestations of SLE patients by preventing the augmentation of apoptosis and, therefore, hindering the autoimmune responses.

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.