[Investigation of the effects and mechanisms of berberine on a mouse model of polycystic ovary syndrome: based on intestinal flora analysis].

Objective: To examine the impact of berberine on polycystic ovary syndrome (PCOS) in mice, and to investigate the effects of berberine on the intestinal flora and the intestinal flora on PCOS. Methods: A mouse model of PCOS was established by administering dehydroepiandrosterone in combination with high fat diet, and the mouse model was given a berberine treatment. The study consisted of a blank control group (C group), a PCOS model group (M group) and a berberine treatment group (T group). During the experiment, the mice were closely monitored through timed body weight measurements and estrous cycle monitoring; intraperitoneal glucose tolerance test and insulin tolerance test were done. Upon completion of the pharmacological intervention, the wet weights of liver, ovary and fat deposits of mice were assessed and subjected to HE staining to confirm the success of PCOS modeling and the efficacy of berberine. Additionally, fecal samples were analyzed for intestinal flora through 16S rRNA analysis. Results: The PCOS model was established successfully, berberine alleviated the disturbance of estrous cycle in mice, and significantly alleviated fat accumulation and metabolic abnormalities of glucose in mice. The cross-sectional area of fat pad cells in T group was (2 858±146) µm², which was significantly lower than that in M group [(9 518±347) µm²], and the difference was statistically significant (P<0.001). The blood glucose levels in T group were significantly lower than those in M group (P<0.05). The composition and structure of intestinal flora in mice of M group with PCOS (compared with C group) and in mice of T group after berberine intervention (compared with M group) were significantly altered. However, alpha diversity did not change significantly among three groups (P>0.05). Conclusion: Berberine could alleviate PCOS by intervening in the alterations of gut microbiota.

Partial agonism by 3alpha,21-dihydroxy-5beta-pregnan-20-one at the gamma-aminobutyric acidA receptor neurosteroid site.

3alpha,21-Dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) and 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P) have full efficacy as allosteric modulators of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to sites on the gamma-aminobutyric acid (GABA) type A receptor complex (GRC). Relative to 3alpha,5alpha-P and 5alpha-THDOC, 3alpha,21-dihydroxy-5beta-pregnan-20-one (5beta-THDOC) has limited efficacy as an allosteric modulator of [35S]TBPS binding. Interactions between 3alpha,5alpha-P, 5alpha-THDOC and 5beta-THDOC were examined to determine whether these neuroactive steroids share a common site for modulation of the GRC. The concentration-response curves for both 3alpha,5alpha-P and 5alpha-THDOC modulation of [35S]TBPS binding to brain and recombinantly derived GRCs are shifted rightward in the presence of various concentrations of 5beta-THDOC. Similarly, 5beta-THDOC modulates GABA-evoked Cl- currents with low efficacy and inhibits the potentiation of GABA-evoked Cl- currents by 3alpha,5alpha-P. Furthermore, behavioral studies reveal that 5beta-THDOC antagonizes 3alpha,5alpha-P-induced loss of the righting reflex in mice at a dose that has no effect alone. These results represent the first demonstration of antagonist-like actions of a neuroactive steroid on the GRCs at levels ranging from the receptor to animal behavior and suggest the existence of partial agonist neurosteroids.

Loreclezole modulates [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding via a distinct site on the GABAA receptor complex.

The allosteric modulation of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam binding was utilized to evaluate the actions of loreclezole at the GABAA receptor complex in the rat brain. Loreclezole was observed to allosterically inhibit the binding of [35S]TBPS in a dose-dependent manner with micromolar potency (IC50 = 1 microM). Loreclezole was found to have an additive effect on neuroactive steroid modulation of [35S]TBPS binding, but merely potentiated the effect of Ro5-4864 (4"-chlorodiazepam) modulation of [35S]TBPS binding. These observations suggest that loreclezole modulates [35S]TBPS binding through a site independent of the neuroactive steroid and Ro5-4864 sites on the GABAA receptor complex. The enhancement of [3H]flunitrazepam binding to the benzodiazepine receptor by loreclezole as well as the effect of loreclezole on CL218872/[3H]flunitrazepam dose-response curves suggest that loreclezole does not act through the benzodiazepine site on the GABAA receptor complex, nor does it selectively modulate benzodiazepine receptor subtypes. The potency of loreclezole as and inhibitor of [35S]TBPS binding in rat brain was regionally dependent and GABA-sensitive. Loreclezole modulation of [35S]TBPS binding showed greater potency and GABA sensitivity in the cerebellum and thalamus when compared to other brain regions such as the cortex, hippocampus and striatum. This finding is consistent with previous reports of the selectivity of loreclezole for GABAA receptor complex's containing beta 2 and beta 3 subunits. These beta subunit isoforms predominate in the cerebellum and thalamus. Collectively the evidence suggests that loreclezole modulates [35S]TBPS and [3H]flunitrazepam binding through a site distinct from benzodiazepine, neuroactive steroid, Ro5-4864 and GABA sites on the GABAA receptor complex.

In vitro reinforcement of hippocampal bursting: a search for Skinner's atoms of behavior.

A novel "in vitro reinforcement" paradigm was used to investigate Skinner's (1953) hypotheses (a) that operant behavior is made up of infinitesimal "response elements" or "behavioral atoms" and (b) that these very small units, and not whole responses, are the functional units of reinforcement. Our tests are based on the assumption that behavioral atoms may plausibly be represented at the neural level by individual cellular responses. As a first approach, we attempted to reinforce the bursting responses of hippocampal units in a highly reduced brain-slice preparation with local micropressure applications of behaviorally reinforcing dopaminergic drugs. The same injections were administered independently of bursting to provide a "noncontingent" control for nonspecific stimulation or facilitation of firing. It was found that the bursting responses of individual CA1 pyramidal neurons may be progressively facilitated in a dose-related manner by response-contingent (but not noncontingent) injections of dopamine itself, the dopamine D1-preferring agonist SKF 82958, the D3-preferring agonist quinpirole, and the D2-like selective agonist (+)-4-propyl-9 hydroxynapthoxazine. These findings support the conclusion that unit bursting responses can be reinforced in vitro in hippocampal slices, and they further suggest that the same dopamine receptor subtypes are involved in both cellular and behavioral operant conditioning. The results thus provide indirect support for Skinner's atoms-of-behavior hypothesis.

In vitro reinforcement of hippocampal bursting by the cannabinoid receptor agonist (-)-CP-55,940.

Involvement of cannabinoid receptors in behavioral reinforcement is suggested by widespread human use of marihuana, although animal tests of cannabinoid reinforcement have produced mixed results. Cannabinoid receptors are found in high density in rat hippocampus and other brain areas. Using the hippocampal-slice preparation, we attempted to demonstrate in vitro reinforcement of CA1 bursting with local micropressure applications of the high-affinity synthetic cannabinoid receptor agonist (-)-CP-55,940. Approximately 60% of the tested neurons showed increased burst activity after a series of brief, burst-contingent applications of (-)-CP-55,940 at pipette concentrations of 5 and 10 microM. Identical microinjections of (-)-CP-55,940 administered independently of cellular activity did not increase and usually suppressed hippocampal bursting. Since general stimulation of CA1 activity by (-)-CP-55,940 can thus be ruled out, we conclude that burst-contingent applications of a cannabinoid receptor agonist can reinforce hippocampal firing in vitro.

A cellular analogue of operant conditioning.

Using the hippocampal-slice preparation, we attempted to model operant conditioning in vitro by reinforcing pyramidal cell bursting responses with local micropressure applications of transmitters and drugs. The same injections were administered independently of bursting to provide a "noncontingent" control for direct pharmacological stimulation or facilitation of firing. The results suggested that the bursting responses of individual CA1 pyramidal neurons may be reinforced in a dose-related manner by response-contingent (but not noncontingent) injections of dopamine and the selective dopamine D2 agonist, N-0923. N-0924, a stereoisomer of N-0923 that is largely devoid of D2-agonist activity, failed to reinforce CA1 bursting. Burst-contingent injections of the excitatory neurotransmitter glutamate also failed to reinforce CA1 bursting; indeed, the glutamate applications (whether contingent or random) reduced the likelihood of bursts while increasing the frequency of solitary spikes. Reinforcement delays exceeding 200 ms largely eliminated the reinforcing efficacy of the D2 agonist N-0437 in CA1 operant conditioning. The results are consistent with the suggestion that the behaviorally reinforcing effects of dopaminergic agents can be modeled in vitro in the hippocampal-slice preparation.

Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences.

Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.

[D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C antagonizes neuromedin C-stimulated amylase release by acini isolated from the rat pancreas.

Two novel neuromedin C analogs [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C and [Leu9-psi-CH2NH-Leu10]neuromedin C, were synthesized by rapid solid phase methods and examined for their abilities to inhibit neuromedin C-stimulated amylase release by isolated rat pancreatic acini. Both analogs significantly inhibited maximally stimulated amylase release by neuromedin C in a dose-dependent manner with maximal inhibition seen at concentrations of 100 and 300 microM of [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C and [Leu9-psi-CH2NH-Leu10]neuromedin C, respectively. The IC50 (concentration required to half-maximally inhibit neuromedin C-stimulated amylase release) was 1.5 microM for [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C compared to a 13.4 microM IC50 for [Leu9-psi-CH2NH-Leu10]neuromedin C. The [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C analog produced a parallel rightward shift in the neuromedin C dose-response curve and Schild plots of the inhibition data gave a slope of 0.969 +/- 0.121 and a pA2 (apparent affinity for the acinar cell receptor in terms of neuromedin C receptor-stimulated amylase release) of 100 nM. While [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C significantly inhibited both neuromedin B- and gastrin releasing peptide-stimulated amylase release, the analog did not inhibit amylase release in response to either cholecystokinin octapeptide, vasoactive intestinal peptide, substance P, carbamylcholine, the Ca2+ ionophore A23187, forskolin, or 8-bromo-cyclic AMP. The results demonstrate that [D-Ala1, Leu9-psi-CH2NH-Leu10]neuromedin C is a potent, specific, and competitive antagonist for neuromedin C and peptides of the gastrin releasing peptide family and may serve as a useful molecule for exploring the physiological role of these peptides.

Chronic administration of a potent cholecystokinin receptor antagonist, L-364,718, fails to inhibit pancreas growth in preweanling rats.

We examined whether endogenous cholecystokinin (CCK) is involved in growth of the preweanling rat pancreas. Twice daily for 14 days, 7-day-old neonatal rats received an oral gavage of either 2.5 mg/kg or 5.0 mg/kg of the potent and specific CCK receptor antagonist L-364,718 (the 2.5 mg/kg dose of antagonist was shown in the present study to abolish totally the pancreas growth-promoting effects of exogenously administered caerulein (CR) in neonatal rats). Control pups received oral gavages of the L-364,718 vehicle alone. The final body weights, pancreas weights, total pancreatic DNA contents, and total pancreatic protein contents did not differ significantly between the 21-day-old control pups and the 21-day-old pups that were pretreated for 14 days with either the low or the high doses of L-364,718. These findings suggest that endogenous CCK is not required for growth of the neonatal rat pancreas.

Evidence for a role of brain thyrotropin-releasing hormone (TRH) on stress gastric lesion formation in rats.

Specific polyclonal antibodies raised against synthetic thyrotropin-releasing hormone (TRH) infused intracerebroventricularly (ICV) significantly decreased gastric lesions induced by cold restraint stress. The antiulcer effect of immunologic blockade of brain TRH was specific. Normal rabbit serum or antibodies raised against somatostatin, alpha-MSH, Leu-enkephalin, gonadotropin-releasing hormone and atrial natriuretic factor were ineffective. These findings suggest that brain TRH may play an important role in experimental stress ulcer formation.

Imipramine prevents gastric lesions induced by centrally administered thyrotropin-releasing hormone (TRH) in rats.

Increasing evidence indicates that thyrotropin-releasing hormone (TRH), and endogenous brain-gut peptide may play a role in experimental ulcerogenesis. Potential interactions between TRH and imipramine (a typical tricyclic antidepressant (TCA] on the development of TRH-induced gastric lesions have not been investigated. Imipramine (0.05, 0.5 and 5 mg/kg, i.p.) dose-dependently inhibited gastric lesion formation induced by intracisternal (i.c.) administration of TRH (1 micrograms). In addition, imipramine (5 mg/kg, i.p.) significantly decreased gastric acid secretion in response to i.c. TRH (1 microgram) in rats with pyloric ligation. These findings suggest the TCAs may be effective drug agents against centrally initiated gastric ulcerations. The mechanism of this response probably involves blockade of cholinergic (muscarinic) and H2 histamine receptors.

Imipramine prevents stress gastric glandular lesions in rats.

Intraperitoneal (i.p.) administration of imipramine dose-dependently inhibited gastric lesions induced by 3 h of cold restraint stress. The high dose of imipramine (5 mg/kg) almost completely abolished gastric lesion formation, and also suppressed acid secretion in rats with pyloric ligation. Intracisternal imipramine (1 microgram) was also protective. These findings suggest that tricyclic antidepressants may play an important role in preventing the deleterious effects of stress on the gut.

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro.

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors.

In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 microM, respectively, could significantly inhibit amylase secretion stimulated by carbachol (P less than 0.01 or 0.05, respectively), and this inhibition by BPP was dose dependent. 45Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated 45Ca outflux. BPP was also capable of displacing the specific binding of [3H]quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (ki 35 nM) than carbachol (Ki 1.8 microM) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant.