Korean J Physiol Pharmacol.  2017 Jul;21(4):429-437. 10.4196/kjpp.2017.21.4.429.

Relaxant and anti-inflammatory effect of two thalidomide analogs as PDE-4 inhibitors in pregnant rat uterus

• Muñoz-Pérez VM
• Fernández-Martínez E
• Ponce-Monter H
• Ortiz MI

• KMID: 2384457
• DOI: http://doi.org/10.4196/kjpp.2017.21.4.429

Abstract

The aim of this study was to evaluate the relaxant and anti-inflammatory effects of two thalidomide analogs as phosphodiesterase-4 (PDE-4) inhibitors in pregnant rat uterus. Uteri from Wistar female rats were isolated at 19 day of pregnancy. Uterine samples were used in functional studies to evaluate the inhibitory effects of the thalidomide analogs, methyl 3-(4-nitrophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4NO2PDPMe) and methyl 3-(4-aminophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4APDPMe), on prostaglandin-F2α (PGF2α)-induced phasic, K⁺-induced tonic, and Ca²âº-induced contractions. Accumulation of cAMP was quantified in uterine homogenates by ELISA. Anti-inflammatory effect was assessed by using ELISA for determination of the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin (IL)-1β, and anti-inflammatory IL-10, from uterine explants stimulated with lipopolysaccharide (LPS). Nifedipine, forskolin and rolipram were used as positive controls where required. Both thalidomide analogs induced a significant inhibition of the uterine contractions induced by the pharmaco- and electro-mechanic stimuli. Nifedipine and forskolin were more potent than the analogs to inhibit the uterine contractility, but these were more potent than rolipram, and 4APDPMe was equieffective to nifedipine. Thalidomide analogs increased uterine cAMP-levels in a concentration-dependent manner. The LPS-induced TNFα and IL-1β uterine secretion was diminished in a concentration-dependent fashion by both analogs, whereas IL-10 secretion was increased significantly. The thalidomide analogs induced utero-relaxant and anti-inflammatory effects, which were associated with the increased cAMP levels as PDE-4 inhibitors in the pregnant rat uterus. Such properties place these thalidomide analogs as potentially safe and effective tocolytic agents in a field that urgently needs improved pharmacological treatments, as in cases of preterm labor.

Keyword

cAMP; Cytokines; Phosphodiesterase-4 inhibitors; Preterm labor; Thalidomide

MeSH Terms

Animals
Colforsin
Cyclic Nucleotide Phosphodiesterases, Type 4
Cytokines
Enzyme-Linked Immunosorbent Assay
Female
Humans
Interleukin-10
Interleukins
Necrosis
Nifedipine
Obstetric Labor, Premature
Phosphodiesterase 4 Inhibitors*
Pregnancy
Rats*
Rolipram
Thalidomide*
Tocolytic Agents
Uterine Contraction
Uterus*
Colforsin
Cyclic Nucleotide Phosphodiesterases, Type 4
Cytokines
Interleukin-10
Interleukins
Nifedipine
Phosphodiesterase 4 Inhibitors
Rolipram
Thalidomide
Tocolytic Agents

Figure

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  Fig. 1 Inhibitory effect of thalidomide analogs, rolipram, forskolin and nifedipine on PGF2α-induced phasic contractions. (a) Concentration-response curves of uterine strips of pregnant rat. Each point in both graphs indicates the mean of 6 experiments (n=6) for thalidomide analogs, rolipram, forskolin and nifedipine; the vertical bars represents the standard error of the mean (SEM). (b) Typical recording of PGF2α-stimulated phasic contractions inhibited by thalidomide analogs in concentration-dependent manner. *Different from rolipram, p<0.05.

• 

  Fig. 2 Inhibitory effect of thalidomide analogs, rolipram, forskolin and nifedipine on KCl-induced phasic contractions. (a) Concentration-response curves of uterine strips of pregnant rat. Each point in both graphs indicates the mean of 6 experiments (n=6) for thalidomide analogs, rolipram, forskolin and nifedipine; the vertical bars represents the standard error of the mean (SEM). (b) Typical recording of KCl-stimulated phasic contractions inhibited by thalidomide analogs in concentration-dependent manner. *Different from rolipram, p<0.05.

• 

  Fig. 3 Inhibitory effect of thalidomide analogs, rolipram, forskolin and nifedipine on Ca2+-induced tonic contractions. (a) Concentration-response curves of uterine strips of pregnant rat. Each point in both graphs indicates the mean of 6 experiments (n=6) for thalidomide analogs, rolipram, forskolin and nifedipine; the vertical bars represents the standard error of the mean (SEM). (b) Typical recording of Ca2+-stimulated tonic contractions inhibited by thalidomide analogs, rolipram, forskolin and nifedipine in concentration-dependent form, *p<0.05.

• 

  Fig. 4 Intracellular cAMP levels induced by thalidomide analogs, rolipram and forskolin in pregnant rat uterus. The production of cAMP induced by forskolin alone (10 µM) and combined with rolipram, 4APDPMe and 4NO2PDPMe was obtained at 100, 300 and 1,000 µM. Each column represents the mean of four experiments (n=4) for analogs, rolipram and forskolin and the vertical bars represent the standard error of the mean (SEM). †Difference vs. Basal; ‡Difference vs. Forskolin; §Difference vs. FSK+Rolipram 1000 µM, p<0.05.

• 

  Fig. 5 Effects of both thalidomide analogs, rolipram and forskolin on the secretion of TNFα induced by LPS in uterine explants of pregnant rat. Explants were cultured in presence of LPS (10 µg/ml), with the PDE-4-inhibitors at 100, 300 and 1,000 µM and with forskolin, FSK (10 µM) for 24 h. Control experiments, basal and DMSO, were run without addition of LPS, or any other compound. Each column represents the mean of four experiments (n=4) for all compounds tested and the vertical bars indicate the standard error of the mean (SEM). †Different vs. basal; ‡Different vs. LPS; §Different vs. LPS+rolipram 1,000 µM, p<0.05.

• 

  Fig. 6 Effects of analogs, rolipram and forskolin on the release of IL-1β induced by LPS in uterine explants of pregnant rat. Explants were cultured in presence of LPS (10 µg/ml), without and with LPS and the PDE-4 inhibitors at 100, 300 and 1,000 µM and forskolin, FSK (10 µM) for 24 h. Control experiments, basal and DMSO, were run without addition of LPS, analogs, rolipram and forskolin. TNFα-levels in supernatants were quantified by a colorimetric ELISA. Each column represents the mean of four experiments (n=4) for all compounds tested and the vertical bars indicate the standard error of the mean (SEM). †Different vs. basal; ‡Different vs. LPS; §Different vs. LPS+rolipram 1,000 µM, p<0.05.

• 

  Fig. 7 Effects of thalidomide analogs, rolipram and forskolin on the secretion of IL-10 induced by LPS in uterine explants of pregnant rat. Explants were cultured with and without LPS (10 µg/ml), and with the three PDE-4 inhibitors at 1000 µM without and with forskolin, FSK (10 µM) for 24 h. Control experiments, basal and DMSO, were run without addition of LPS, analogs, rolipram and forskolin. Each column represents the mean of four experiments (n=4) for all compounds tested and the vertical bars indicate the standard error of the mean (SEM). †Different vs. basal; ‡Different vs. LPS; §Different vs. rolipram 1,000 µM; #Different vs. LPS+rolipram 1,000 µM, p<0.05.

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