[Treatment of Chronic Endometritis by Penning Granule: a Clinical Randomized Controlled Trial].

Objective To observe the mechanism of Penning Granule ( PG) for treating chronic endometritis (CE). Methods Totally 38 CE patients in line with inclusive criteria were assigned to the PG group (23 cases) and the control group (15 cases) according to random digit table. PG (consisting of sargentgloryvine stem, Herba Patriniae, Hedyotis Diffusa, red peony root, Angelica Sinensis, prepared mastiche, prepared myrrh, common burreed tuber, zedoary root, Spina Gleditsiae, Danshen root, Radix Bupleuri, asarum , Astragalus, Pangolin) was administered to patients in the PG group, 105 g each time, twice per day for 4 successive weeks. Those in the control group took Levofloxacin (0.5 g, once per day) and Metronidazole Tablet (0. 5 g, twice daily for 1 successive week). They were followed-up for half a year after ending treatment. The morphological changes of uterine endometrium were observed by hyst- eroscopy before and after treatment, and endometrium biopsy performed at fixed position. The expres- sions of mucin-1 (MUC-1 ) and hypoxia induced facter-1 α (HIF-1α) in inflammatory endometrium were analyzed. The clinical effects were compared between PG and antibiotics from improvement of clinical symptoms, hysteroscopic manifestations, pathological analyses, and molecular levels. Results The markedly effective rate of clinical symptoms in the PG group was 91. 3% (21/23) , higher than that of the control group [60. 0% (9/15) ; P <-0. 05]. The plasma cell CD38 infiltration in endometrial stroma of the PG group were significantly decreased, showing better effect than antibiotics in the control group (P < 0.05). The positive expression of MUC-1 was increased and the expression of HIF-1 α was decreased in the PG group, showing better effect than antibiotics in the control group (P <0. 05). Conclusion PG showed obvious effects for treating chronic endometritis , and it was superior to that of antibiotics alone.

Agonist function of the recombinant alpha 4 beta 3 delta GABAA receptor is dependent on the human and rat variants of the alpha 4-subunit.

1. It is known that the alpha(4)-subunit is likely to occur in the brain predominantly in alpha(4)beta(3)delta receptors at extrasynaptic sites. Recent studies have revealed that the alpha(1)-, alpha(4)-, gamma(2)- and delta-subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA(A) receptors containing human (H) and rat (R) alpha(1)/alpha(4)-, beta(2)/beta(3)- and gamma(2S)/delta-subunits in Xenopus oocytes using the two-electrode voltage-clamp technique. 2. Both H alpha(1)beta(3)delta and H alpha(4)beta(3)gamma(2S) receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the alpha(4)beta(3)delta combination was more sensitive to agonist action than the alpha(4)beta(3)gamma(2S) receptor, we observed extremely small GABA- and pentobarbital-activated currents at the wild-type H alpha(4)beta(3)delta receptor. However, GABA and pentobarbital activated the wild-type R alpha(4)beta(3)delta receptor with high potency (EC(50) = 0.5 +/- 0.7 and 294 +/- 5 micromol/L, respectively). 3. Substituting the H alpha(4) subunit with R alpha(4) conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC(50) and increased I(max). When the H alpha(4) subunit was combined with the R beta(3) and R delta subunit in a heteropentameric form, the amplitude of GABA- and pentobarbital-activated currents increased significantly compared with the wild-type H alpha(4)beta(3)delta receptor. 4. Thus, the results indicate that the R alpha(4)beta(3)delta, H alpha(1)beta(3)delta and H alpha(4)beta(3)gamma(2S) combinations may contribute to functions of extrasynaptic GABA(A) receptors. The presence of the R alpha(4) subunit at recombinant GABA(A) receptors containing the delta-subunit is a strong determinant of agonist action. The recombinant H alpha(4)beta(3)delta receptor is a less sensitive subunit composition in terms of agonist activation.

Neurosteroids 3beta, 20 (R/S)-pregnandiols decrease offset rate of the GABA-site activation at the recombinant GABA A receptor.

Neurosteroids directly modulate ligand gated ion channels such as GABA A receptors. Two such molecules, 3beta-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABA A receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5alpha-pregnan-3beta,20(S)-diol (UC1019), 5beta-pregnan-3beta, 20(R)-diol (UC1020) and PS on the activation onset and offset times of the recombinant GABA A receptor (rat alpha1beta2gamma2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 microM GABA for 10 s, followed by a 80 s washout period. PS (> ir =0.3 microM) moderately increased the slow onset rate (k(on-S)) of GABA-response. PS had no significant effects on the fast onset rate (k(on-F)). UC1019 and UC1020 decreased the k(on-S) of the GABA-response in a concentration-dependent manner with no significant effects on the k(on-F). Like PS, UC1019 and UC1020 decreased the slow offset rates (k(off-S)). In addition, PS increased the fast offset rate (k(off-F)) in a concentration-dependent manner, while UC1019 and UC1020 decreased k(off-F). The EC50 of PS to increase k(off-F) was calculated as 0.47+/-0.1 microM. The corresponding IC50 values of UC1019 and UC1020 to decrease k(off-F) were 5.0+/-0.5 microM and 8.4+/-0.9 microM, respectively. These results suggest differential actions of PS and 3beta, 20(R/S)-pregnandiols on the offset time course of GABA-site activation.

A comparison of the pharmacological properties of recombinant human and rat alpha(1)beta(2)gamma(2L) GABA(A) receptors in Xenopus oocytes.

In the present study, we compared the pharmacology, particularly neurosteroid modulation of the GABA(A) receptor, between human and rat alpha(1)beta(2)gamma(2)(L) GABA(A) receptors and between human receptors containing the long (L) and short (S) forms of the gamma(2)-subunit. We observed that maximum responses to GABA were significantly higher with the human alpha(1)beta(2)gamma(2)(L) receptor compared with the rat receptor. In terms of neurosteroid modulation, increases in the EC(15) response to GABA induced by 3alpha-OH-5beta-pregnan-20-one (3alpha5betaP), 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) and 5alpha-pregnane-3alpha,20beta-diol (3alpha5alpha-diol) were significantly greater for the rat compared with the human receptor. Responses to 30 micromol/L GABA were inhibited by 3beta-OH-5alpha-pregnan-20-one (UC1010) and 5beta-pregnan-3beta,20(R)-diol (UC1020) to a greater degree for human and rat receptors, respectively. Responses to GABA + 3alpha5alphaTHDOC were inhibited by 5alpha-pregnan-3beta,20(S)-diol (UC1019) and pregnenolone sulphate to a greater degree for human and rat receptors, respectively. The GABA dose-response curves for human alpha(1)beta(2)gamma(2)(S) and alpha(1)beta(2)gamma(2)(L) receptors were identical. However, the maximum GABA-evoked current, the direct gating effect of pentobarbital and the allosteric potentiation of the GABA EC(15) response by 3alpha5alphaTHDOC and 3alpha5betaP were significantly higher with alpha(1)beta(2)gamma(2)(S) than alpha(1)beta(2)gamma(2)(L) receptors. Inhibition of the response to 30 micromol/L GABA by UC1010 and UC1020 was greater for a(1)beta(2)gamma(2)(L) and alpha(1)beta(2)gamma(2)(S) receptors, respectively. Inhibition of responses to 3alpha5alphaTHDOC + GABA by UC1019 and UC1010 was significantly higher for alpha(1)beta(2)gamma(2)(L) receptors. In conclusion, the site of activation by GABA and neurosteroid modulation differ between human and rat alpha(1)beta(2)gamma(2)(L) receptors, as well as between human receptors containing the L and S splice variants of the gamma(2)-subunit.

3Beta-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABA(A) receptor through different channel property.

3Beta-hydroxysteroids are pregnenolone sulfate-like GABA(A) receptor antagonists. The aim of the current study was to compare the functional differences between 3beta-hydroxysteroids and pregnenolone sulfate to inhibit GABA(A) receptors expressed in Xenopus oocytes. Recombinant rat GABA(A) receptors encoding wild type alpha1 beta2 gamma2L receptor, mutant alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L receptors were examined using a two-electrode voltage-clamp technique. A homologous mutation of the residue at 2'position closest to the cytoplasmic end of the M2 helix to serine on both alpha1 and beta2 subunit, alpha1V256S and beta2A252S, reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly in the alpha1V256S beta2 gamma2L receptor (P<0.05), whereas it decreased moderately in the alpha1 beta2A252S gamma2L receptor. We found that 5alpha-pregnan-3beta, 20(S)-diol (UC1019) and 5beta-pregnan-3beta, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3beta-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC50s of 0.20+/-0.07 microM; 1.88+/-0.32 microM and 2.58+/-0.58 microM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L receptors (P<0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant alpha1V256S beta2 gamma2L receptor. Pregnenolone sulfate promoted slow desensitization with prolonged GABA application in a dose-dependent manner in the wild type receptor, but not mutant receptors. On the contrary, UC1019 and UC1020 (< or = 20 microM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABA(A) receptor inhibition by pregnenolone sulfate, but not 3beta-hydroxysteroids, was dependent on desensitization kinetics of the Cl- channels. A point mutation at M2 helix of the beta2-subunit (beta2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABA(A) receptors without affecting the inhibitory properties of 3beta-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3beta-hydroxysteroids the coincident channel property at the GABA(A) receptor.

Neurosteroid modulation of recombinant rat alpha5beta2gamma2L and alpha1beta2gamma2L GABA(A) receptors in Xenopus oocyte.

GABA(A) receptors containing alpha(5)-subunit have an important role in cognitive function. As the agonistic effect of 3alpha-hydroxy ring-A reduced steroids depends on subunit combinations of the GABA(A) receptor, the antagonistic effect of pregnenolone sulfate and 3beta-hydroxypregnane steroids may vary between alpha(5)-subunit and alpha(1)-subunit containing receptors. We investigated the effect of agonist and antagonist steroids in the recombinant rat alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors expressed in Xenopus oocytes using a two electrodes voltage-clamp technique. We did not find any significant difference in potency and efficacy of GABA response between alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors. Compared to the alpha(1)beta(2)gamma(2L) receptor, a significantly lower degree of desensitization was observed in the alpha(5)beta(2)gamma(2L) receptor. In addition, the potencies of 3alpha-OH-5alpha-pregnan-20-one (3alpha5alphaP), 5alpha-pregnan-3alpha,21-diol-20-one (3alpha5alphaTHDOC) and 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) to enhance GABA response were significantly higher in the alpha(5)beta(2)gamma(2L) receptor, whereas their efficacies remained unchanged between two receptors. In either receptor, the efficacy of 3alpha5alphaTHDOC was significantly higher than 3alpha5alphaP and 3alpha5alphaADL. The efficacies of 5beta-pregnan-3beta,21-diol-20-one(UC1015) and 5alpha-pregnan-3beta,20alpha-diol(UC1019) to inhibit 30 microM GABA response, and the efficacies of 3beta-OH-5beta-pregnan-20-one (UC1014) and 5beta-pregnan-3beta, 20beta-diol (UC1020) to inhibit 3 microM 3alpha5alphaTHDOC+3 microM GABA response were higher in the alpha(5)beta(2)gamma(2L) receptor compared to the alpha(1)beta(2)gamma(2L) receptor. The potencies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response and the 3alpha5alphaTHDOC+GABA response did not vary between two receptors. Interestingly, the potencies and efficacies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response were positively correlated to their potencies and efficacies to inhibit the 3alpha5alphaTHDOC+GABA response. Results from the current study revealed a different modulation pattern by neurosteroids between the alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptor.

Neuroactive steroid effects on cognitive functions with a focus on the serotonin and GABA systems.

This article will review neuroactive steroid effects on serotonin and GABA systems, along with the subsequent effects on cognitive functions. Neurosteroids (such as estrogen, progesterone, and allopregnanolone) are synthesized in the central and peripheral nervous system, in addition to other tissues. They are involved in the regulation of mood and memory, in premenstrual syndrome, and mood changes related to hormone replacement therapy, as well as postnatal and major depression, anxiety disorders, and Alzheimer's disease. Estrogen and progesterone have their respective hormone receptors, whereas allopregnanolone acts via the GABA(A) receptor. The action of estrogen and progesterone can be direct genomic, indirect genomic, or non-genomic, also influencing several neurotransmitter systems, such as the serotonin and GABA systems. Estrogen alone, or in combination with antidepressant drugs affecting the serotonin system, has been related to improved mood and well being. In contrast, progesterone can have negative effects on mood and memory. Estrogen alone, or in combination with progesterone, affects the brain serotonin system differently in different parts of the brain, which can at least partly explain the opposite effects on mood of those hormones. Many of the progesterone effects in the brain are mediated by its metabolite allopregnanolone. Allopregnanolone, by changing GABA(A) receptor expression or sensitivity, is involved in premenstrual mood changes; and it also induces cognitive deficits, such as spatial-learning impairment. We have shown that the 3beta-hydroxypregnane steroid UC1011 can inhibit allopregnanolone-induced learning impairment and chloride uptake potentiation in vitro and in vivo. It would be important to find a substance that antagonizes allopregnanolone-induced adverse effects.

Protons inhibit Cl- conductance by direct or allosteric interaction with the GABA-binding site in the rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptor.

Functional roles of external pH on the Cl- conductance were examined on Xenopus oocytes expressing rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptors. Acidic pH inhibited GABA-response in a reversible and concentration-dependent manner, significantly increasing the EC50 without appreciably changing the slope or maximal currents induced by GABA in the alpha1beta2gamma2L and alpha1beta2 receptors. In contrast, protonation did not influence the pentobarbital-gated currents in the alpha1beta2gamma2L receptors, suggesting that protons do not modulate channel activity by directly affecting the channel gating process. Protons competitively inhibited the bicuculline-induced antagonism on GABA in the alpha1beta2gamma2L receptors. The data support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA-binding site.

Isoallopregnanolone; an antagonist to the anaesthetic effect of allopregnanolone in male rats.

The interaction of isoallopregnanolone (3 beta-OH-5 alpha-pregnan-20-one) on allopregnanolone (3 alpha-OH-5 alpha-pregnan-20-one) induced anaesthesia was studied in male rats using burst suppression of 1 s ("silent second") with an electroencephalographic-threshold method. The i.v. administration of isoallopregnanolone was varied in relation to induction of "silent second". Pre-treatment with isoallopregnanolone (12.5-50 mg/kg iv) 2 min prior to the threshold test gave an increase in the threshold dose of allopregnanolone (ANOVA df(3;36), F=13.61, P<0.001), which was dose dependent (r=0.73, b [slope]=0.08, df=38, P<0.001). After isoallopregnanolone pre-treatment, but not in the controls, anaesthesia time was positively related to the dose of allopregnanolone (r=0.52, b=1.72, df=28, P<0.01). Anaesthesia times were not influenced by a corresponding administration of isoallopregnanolone immediately after induction of "silent second". When allopregnanolone and isoallopregnanolone were infused together at molar ratios of 1:1, 1:1.23, 1:1.43, a linear increase of the threshold doses of allopregnanolone was seen in relation to the dose of isoallopregnanolone (r=0.86, b=0.40, df=8, P<0.01). Thus isoallopregnanolone can antagonise the anaesthetic action of allopregnanolone.

Ovarian hormone effects on 5-hydroxytryptamine(2A) and 5-hydroxytryptamine(2C) receptor mRNA expression in the ventral hippocampus and frontal cortex of female rats.

Alterations in female gonadal hormones are associated with anxiety and mood changes. The aim of the present study was to determine influences of chronic gonadal hormone supplementation on 5-HT(2A) and 5-HT(2C) receptor mRNA levels in the ventral hippocampus and the frontal cerebral cortex. Ovariectomized adult female Sprague-Dawley rats (n=37) received implantation of subcutaneous pellets containing different dosages of 17beta-estradiol alone or in combination with progesterone, or placebo pellets, for 2 weeks. Serotonin receptor mRNA levels were analyzed by in situ hybridization in the ventral hippocampus and 5-HT(2A) receptor mRNA also in the frontal cortex. Estradiol treatment in combination with low-dose progesterone increased 5-HT(2A) receptor mRNA by 43% in the CA2 region of the ventral hippocampus, while estradiol combined with high-dose progesterone increased the expression of this gene by 84% in ventral CA1. 5-HT(2A) mRNA expression in the frontal cortex was not influenced by hormone manipulation. 5-HT(2C) receptor gene expression was in the ventral hippocampus decreased in the CA2, ventral CA1 and the subiculum subregions by high-dose estradiol treatment (8-20% decreases). Effects on mood by gonadal hormones can be mediated, at least partly, through influences on 5-HT(2A) and 5-HT(2C) receptor expression.