Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosome Repairs Endometrial Epithelial Cells Injury Induced by Hypoxia via Regulating miR-663a/CDKN2A Axis.

Aim: Thin endometrium remains a severe clinical challenge with no effective therapy to date. We aimed at exploring the role and molecular mechanism of human umbilical cord mesenchymal stem cell- (hucMSC-) derived exosomes (hucMSC-Ex) in repairing hypoxic injury of endometrial epithelial cells (EECs). Methods: Exosomes were harvested from the conditioned medium of hucMSC and characterized using western blot, transmission electron microscopy (TEM), flow cytometry, and nanoparticle tracking analysis (NTA). EECs were subjected to hypoxic conditions before cocultured with hucMSC-Ex. Cell viability, apoptosis, and migration were determined with CCK-8, flow cytometry, and wound healing assay, respectively. Apoptosis/EMT-related proteins were detected by western blot. The miRNA profiling was determined by RNA sequencing. The expression of miR-663a and CDKN2A was measured by qRT-PCR. MiR-663a in EECs was overexpressed by transfecting with miR-663a mimics. Results: Mesenchymal stem cells (MSCs) markers CD73, CD90, and CD106 were positively expressed in hucMSCs. Exosome isolated from hucMSC expressed CD63 and TSG101, and were 100-150 nm in diameter. HucMSC-Ex promoted cell proliferation inhibited by hypoxia. And hucMSC-Ex also inhibited hypoxia-induced apoptosis, migration, and EMT of EECs by upregulating the expression of Bcl-2 and E-cadherin and downregulating Bax and N-cadherin levels. Further, bioinformatics research found that hucMSC-Ex coculture can significantly upregulate the expression of miR-663a and decrease the expression of CDKN2A in hypoxia-induced EECs. Furthermore, miR-663a overexpression inhibited CDKN2A expression and increased the expression of Bcl-2 and E-cadherin in hypoxia-induced EECs. Conclusions: HucMSC-Ex promoted cell proliferation, inhibited cell apoptosis, migration, and EMT in hypoxia-induced EECs, thereby alleviating hypoxia-induced EECs injury, which may be related to its regulation of miR-663a/CDKN2A expression. Our study indicated that hucMSC-Ex might benefit for repairing thin endometrium.

Differential effects of cholecystokinin (CCK-8) microinjection into the ventrolateral and dorsolateral periaqueductal gray on anxiety models in Wistar rats.

Cholecystokinin (CCK) is one of the main neurohormone peptide systems in the brain, and a major anxiogenic mediator. The periaqueductal gray (PAG) is a key midbrain structure for defensive behaviors, which could include anxiety, fear, or even panic. The CCK system has wide distribution in the PAG, where the dorsolateral region (DL) participates in active defensive behavior and the ventrolateral region (VL) in passive defensive behavior. The aim of this study was to assess the effect of CCK-8 microinjection into DL-PAG or VL-PAG on anxiety-like behavior through two tests: elevated plus maze (EPM) and defensive burying behavior (DBB). CCK-8 (0.5 and 1.0 µg/0.5 µL) presently microinjected into the DL-PAG produced an anxiogenic-like effect on the EPM evidenced by decreasing the time spent/number of entries in open arms compared to vehicle group. Additionally, the latency to burying decreased and burying time increased on the DBB test. Contrarily, CCK-8 microinjected into the VL-PAG resulted in greater open-arm time and more open-arm entries compared to the vehicle-microinjected group. The results on the DBB test confirmed an anxiolytic-like response of CCK-8 into the VL-PAG. In conclusion, CCK-8 microinjected into DL-PAG produced anxiety-like behavior on EPM, and for first time reported on DBB. Contrarily, CCK-8 microinjected into the VL-PAG reduced anxiety-like behavior also for first time reported using both behavioral models EPM and DBB.

C-type natriuretic peptide applied to the brain enhances exocrine pancreatic secretion through a vagal pathway.

C-type natriuretic peptide (CNP) is the major natriuretic peptide in the brain and its mRNA has been reported in the central nervous system, which supports local synthesis and its role as a neuromodulator. The aim of the present work was to study the effect of centrally applied CNP on pancreatic secretion. Rats were fitted with a lateral cerebroventricular cannula one-week before secretion studies. The central administration of CNP dose-dependently enhanced pancreatic fluid and protein output. CNP response was diminished by atropine and hexamethonium, but it was abolished by vagotomy. Neither adrenergic antagonists nor the administration of (D-p-Cl-Phe(6),Leu(17))-vasoactive intestinal peptide (VIP antagonist) or N(omega) Nitro-L arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor) affected CNP response. The effect induced by CNP was mimicked by 8-Br-cGMP but not by c-ANP-(4-23) amide (selective agonist of the natriuretic peptide receptor C). Furthermore, CNP interacted with cholecystokinin (CCK) and secretin in the brain to modify pancreatic secretion. Present findings show that centrally applied CNP enhanced pancreatic secretion through a vagal pathway and suggest that CNP response is mediated by the activation of natriuretic peptide guanylyl cyclase coupled receptors in the brain.

Electrophysiological responses of nucleus tractus solitarius neurons to CCK and gastric distension in newborn lambs.

The effect of cervical vagus nerve stimulation, gastric distension and CCK-8S administration was studied on the activity of 120 neurons located in the nucleus tractus solitarius (NTS) of anesthetized newborn lambs. One hundred cells responded to the three different inputs. The distribution of the cells in the NTS was from 3 mm rostral to 3 mm caudal to the obex, the major responsive cells being located at the level of the obex. Neurons were either excited or inhibited by gastric distension and CCK-8S, and the responses to these two stimuli were always in the same direction. A small number of cells responded to gastric distension and CCK-8S but not to vagus nerve stimulation. Injection of the CCK-A receptor antagonist 2-NAP abolished both the responses to CCK-8S and to gastric distension. The results are consistent with the idea that CCK-8S acts directly on vagal mechanoreceptive endings in the gastric corpus close to duodenum. These results from lambs may reflect the pathway by which gastric distension and peripheral CCK-8S modulate NTS cells activity during colostrum ingestion, which could in turn activate structures related to learning and memory processes involved in the development of mother preference.

Systemic and intra-dorsal periaqueductal gray injections of cholecystokinin sulfated octapeptide (CCK-8s) induce a panic-like response in rats submitted to the elevated T-maze.

The neuropeptide cholecystokinin (CCK) has been implicated in fear and anxiety. CCK is found in the CNS in several molecular forms such as the tetrapeptide (CCK-4) and, mainly, the sulfated octapeptide (CCK-8s) fragments. Administration of CCK-4 induces panic attacks in humans and increases the expression of different anxiety-related behaviors in laboratory animals. The effects of CCK-8s on fear and anxiety are less straightforward and seem to be influenced, among other factors, by the route of the peptide administration and the animal model employed. In other to further investigate the role of CCK-8s in fear and anxiety, in the present study we analyzed the effect of CCK-8s in male Wistar rats submitted to the elevated T-maze. This animal model of anxiety was developed in order to separate generalized anxiety (inhibitory avoidance) and panic-like (escape) responses in the same rat. The effect of CCK-8s in this test was also investigated after injection of the peptide into the dorsal periaqueductal gray (DPAG). This brainstem area is rich in CCK receptors and has consistently been implicated in the mediation of fear and anxiety responses. The results showed that both the intraperitoneal and intra-DPAG injections of CCK-8s potentiated one-way escape behavior, suggesting a panicogenic action. In contrast, the injection of the CCK2 receptor antagonist CR2945 inhibited the expression of this behavior, a panicolytic-like effect. Therefore, the elevated T-maze, in contrast to other animal models of anxiety, can detect the anxiety-eliciting effects of CCK-8s both after its systemic and central administration. Also, the results provide further evidence about the involvement of a CCK-mediated mechanism within the DPAG in the regulation of panic-related defensive behaviors.

Anxiogenic effect of cholecystokinin in the dorsal periaqueductal gray.

Systemic administration of cholecystokinin (CCK) fragments produces anxiogenic effects. The dorsal periaqueductal gray (dPAG) has been related to anxiety and panic reactions. The objective of this study was to investigate a possible anxiogenic effect of CCK-8 microinjected into the dPAG. At 10 min after the last microinjection (0.5 microl) into the dPAG male Wistar rats (N=7-17) were tested in the elevated plus-maze, an animal model of anxiety. The following treatments were tested alone or in combination: sulfated CCK-8 (CCK-8s, 0.5-1 microg), PD 135158 (N-methyl-D-glucamine, 0.1 microg), a CCK-2 receptor antagonist, lorglumide (0.1-0.3 microg), a CCK-1 receptor antagonist. In addition, Fos immunohistochemistry was performed in rats (n=3-4) treated with CCK-8s (1 microg) alone or in combination with PD 135158 (0.1 microg). CCK-8s produced anxiogenic-like effect, decreasing the percentage of time spent in open arm (saline=30.3+/-6.6, CCK 0.5 microg=15.2+/-1.8; CCK 1 microg=14.6+/-2.1). This effect was prevented by pretreatment with PD 135158, but not by lorglumide. CCK-8s injected into the dPAG induced Fos immunoreactivity in several brain areas related to defensive behavior, including the PAG, median, and dorsal raphe nuclei, superior colliculus, lateral septal nuclei, medial hypothalamus, and medial amygdala. This effect was also prevented by pretreatment with PD 135,158. These results suggest that CCK-8s, acting on CCK-2 receptors, may modulate anxiety reactions in the dPAG.

Cholecystokinin modulation of apomorphine- or amphetamine-induced stereotypy in rats: opposite effects.

Stereotyped behavior can be induced by the dopamine agonist apomorphine or by the releasing agent amphetamine. Cholecystokinin influence on dopamine-mediated behaviors has been extensively studied but a real controversy remains. Our purpose was to further characterize the dopamine-cholecystokinin interaction in apomorphine- and amphetamine-induced stereotyped behavior using sulphated cholecystokinin octapeptide (CCK8) and cholecystokinin tetrapeptide (CCK4) treatments. The results showed that CCK8 decreases apomorphine-induced stereotyped behavior and CCK4 has no effect. CCK4 and CCK8 increased the amphetamine-induced stereotyped behavior; CCK4 was more effective. The results confirm the opposite modulation of apomorphine or amphetamine-induced stereotyped behavior by CCK. These data suggest that this modulation is mediated by both CCK receptors on apomorphine-induced and only by CCK(2) receptors on amphetamine-induced stereotyped behavior.

Enhanced intestinal motor response to cholecystokinin in post-Nippostrongylus brasiliensis-infected rats: modulation by CCK receptors and the vagus nerve.

The jejunal inflammation induced in rats by the nematode Nippostrongylus brasiliensis is followed by intestinal neuroimmune alterations including mast cell hyperplasia and nerve remodelling. On the other hand, cholecystokinin (CCK) plays a pivotal role in the regulation of intestinal motility. The aim of this study was to determine whether the intestinal motor response to CCK is altered 30 days after infection by N. brasiliensis. Thus, CCK-8 (50 microg kg(-1) intraperitoneally) disrupted the pattern of jejunal migrating myoelectric complexes for a longer time in postinfected rats (95.5 +/- 3.5 min) than in controls (48.1 +/- 5.1 min). This enhanced jejunal response was also found after oral administration of the potent releaser of endogenous CCK, soybean trypsin inhibitor. In contrast, no alteration of the inhibition of colonic motility by CCK administration was observed. The increased responsiveness of jejunal motility to CCK persisted after mast cell stabilisation or depletion but was prevented by atropine, devazepide and L-365260 (CCK-A and CCK-B receptor antagonists, respectively) and vagotomy. These results indicate that neuroimmune alterations after N. brasiliensis infection lead to an increased intestinal motility response to CCK that involves a cholinergic mediation, a vagal pathway and alterations in intestinal CCK-A and CCK-B receptors.

Interaction of serotonin and cholecystokinin in the lateral parabrachial nucleus to control sodium intake.

Serotonin [5-hydroxytryptamine (5-HT)] and CCK injected into the lateral parabrachial nucleus (LPBN) inhibit NaCl and water intake. In this study, we investigated interactions between 5-HT and CCK into the LPBN to control water and NaCl intake. Male Holtzman rats with cannulas implanted bilaterally in the LPBN were treated with furosemide + captopril to induce water and NaCl intake. Bilateral LPBN injections of high doses of the 5-HT antagonist methysergide (4 microg) or the CCK antagonist proglumide (50 microg), alone or combined, produced similar increases in water and 1.8% NaCl intake. Low doses of methysergide (0.5 microg) + proglumide (20 microg) produced greater increases in NaCl intake than when they were injected alone. The 5-HT(2a/2c) agonist 2,5-dimetoxy-4-iodoamphetamine hydrobromide (DOI; 5 microg) into the LPBN reduced water and NaCl intake. After proglumide (50 microg) + DOI treatment, the intake was not different from vehicle treatment. CCK-8 (1 microg) alone produced no effect. CCK-8 combined with methysergide (4 microg) reduced the effect of methysergide on NaCl intake. The data suggest that functional interactions between 5-HT and CCK in the LPBN may be important for exerting inhibitory control of NaCl intake.

A possible interaction between CCKergic and GABAergic systems in the rat brain.

Cholecystokinin sulfated octapeptide (CCK-8S) was given to rats i.p. at single doses of 10 and 100 nmol/kg, respectively. It produced a modification in GABA levels in several areas of the rat brain. After 30 min of injection, the lower dose (10 nmol/kg) increased GABA levels in striatum by 31% (P<0.05). The higher dose (100 nmol/kg) enhanced GABA levels either in hippocampus by 78% (P<0.05) or in frontal cerebral cortex by 81% (P<0.05) and decreased in olfactory bulbs by 57% (P<0.01). Thus, these results show that systemic injection of CCK-8S, produced regional specific changes on GABA levels in brain, and these effects were dose-dependent. Systemic pretreatment with the CCK(B) receptor antagonist, PD 135,158, 1 mg/kg i.p., on the endogenous levels of GABA in certain regions was also studied. The selective CCK(B) receptor antagonist, PD 135,158, did not have an effect per se on the endogenous levels of GABA but prevents the action induced by the neuropeptide. We suggest that the action of CCK may be mediated via a selective action on the CCK(B) receptor subtypes.

Administration of cholecystokinin sulphated octapeptide (CCK-8S) induces changes on rat amino acid tissue levels and on a behavioral test for anxiety.

1. The effect of the intraperitoneal administration of cholecystokinin sulphated octapeptide (CCK-8S) (10 nmol/kg i.p.) on endogenous levels of several amino acids in five areas of the rat brain was analyzed. The olfactory bulb, hypothalamus, hippocampus, cerebral frontal cortex, and corpus striatum were evaluated. In addition, the effects of CCK-8S and PD 135,158 (1 mg/kg), a selective CCK(B) antagonist, on the performance of rats submitted to a dark/light transition test were also studied. 2. Upon administration of CCK-8S, the concentration of glutamate was reduced (27%) in the olfactory bulb. The same was observed when the levels of glycine (31%) or alanine (43%) were determined. No significant effects were produced by CCK-8S on cortical and hypothalamic levels. In the hippocampus, the concentration of both glutamate (27%) and taurine (29%) were reduced, whereas the levels of GABA in the striatum (29%) were increased. 3. After a single injection of CCK-8S, the time spent by the rats in the illuminated site of the dark/light transition test box, was not changed. On the contrary, the administration of PD 135,158 increased the time spent in the lighted compartment. 4. These results show that systemic administration of CCK-8S produced regional specific changes in brain amino acids, without producing any significant behavioral modification in the rat exposed to a dark/light box. In contrast, the selective CCKB receptor antagonist, PD 135,158, induces anxiolytic-like action in an animal model of anxiety.

Modulation of apomorphine-induced stereotyped behavior by cholecystokinin.

1. The goal was to verify if central or peripheral sulphated cholecystokinin octapeptide (CCK8) injections can modulate apomorphine (APO)-induced stereotyped behavior. Experiments were designed to determine the involvement of cholecystokinin receptor subtypes as well. 2. Animals which received CCK8 (0.0725, 0.145 and 14.5 nmol, icv) showed a significant (p < 0.05) decrease in APO (0.6 mg/kg, sc)-induced stereotyped behavior. 3. No other statistically significant difference was observed among groups. Since ip CCK8 (1.16 or 2.32 nmol/kg) injections did not interfere with APO-induced stereotypy, the results suggest that the CCK8 modulatory effects have a central action. 4. The results also suggest that the effects of icv CCK8 were not due to the stimulation of CCK8 receptors alone since central CCK4 (14.5 or 29.0 nmol) injections did not interfere with the expression of stereotypy.

Intracerebroventricular administration of cholecystokinin reduces stereotypy in dopamine-supersensitive rats.

Cholecystokinin (CCK-8) coexists with dopamine in some neurons and modulates dopaminergic neurotransmission. In the present study we determined the effect of CCK-8 on stereotyped behavior in supersensitive dopaminergic system. Adult male Wistar rats, weighing 200-250 g, were used. Dopaminergic supersensitivity was induced by long-term haloperidol (HAL) treatment (30 days: 1.0 mg/kg twice a day). Seventy-two hours after HAL withdrawal animals received CCK-8 (14.5 nmol/5 microliters) or saline intracerebroventricularly (icv) before being tested for apomorphine (APO, 0.6 mg/kg, sc)-induced stereotyped behavior. Experimental groups were: long-term HAL-treated rats that received saline (HSAL, N = 9) or CCK-8 (HCCK, N = 11) icv and long-term saline-treated rats that received CCK-8 (SCCK, N = 9) or saline (SSAL, N = 8) icv. As expected, HSAL rats showed statistically significant higher stereotypy scores than SSAL rats (42 +/- 1.7 vs 31 +/- 1.6; P < 0.05) and CCK-8 significantly reduced stereotyped behavior in supersensitive rats (42 +/- 1.7 vs 37 +/- 1.5; P < 0.05). These results show that CCK-8 icv reduces stereotypy in dopaminergic-supersensitive rats, and suggest that the dopamine supersensitivity phenomenon can be modulated by CCK-8.

Intracerebroventricular administration of cholecystokinin reduces stereotypy in dopamine supersensitive rats

Cholecystokinin (CCK-8) coexists with dopamine in some neurons and modulates dopaminergic neurotransmission. In the present study we determined the effect of CCK-8 on stereotyped behavior in supersensitive dopaminergic system. Adult male Wistar rats, weighing 200-250 g, were used. Dopaminergic supersensitivity was induced by long-term haloperidol (HAL) treatment (30 days: 1.0 mg/kg twice a day). Seventy-two hours after HAL withdrawal animals received CCK-8 (14.5 nmol/5 µl) or saline intracerebroventricularly (icv) before being tested for apomorphine (APO, 0.6 mg/kg, sc)-induced stereotyped behavior. experimental groups were: long-term HAL-treated rats that received saline (HSAL, N = 9) or CCK-8 (HCCK, N = 11) icvand long-term saline-treated rats that received CCK-8(SCCK,N = 9) or saline (SSAL, N = 8) icv. As expected, HSAL rats showed statistically significant higher stereotypy scores than SSAL rats (42 + or - 1.7 vs 31 + or - 1.6; P<0.05) and CCK-8 icv reduces stereotypy in dopaminergic-supersensitive rats, and suggest that the dopamine supersensitivity phenomenon can be modulated by CCK-8

Cholecystokinin-8 increases K(+)-evoked [3H] gamma-aminobutyric acid release in slices from various brain areas.

[3H] gamma-Aminobutyric acid (GABA) release was studied in rat brain slices in the absence or presence of cholecystokinin-8 (CCK-8). [3H]GABA release under the conditions used was Ca(2+)-dependent and insensitive to the presence of the glial uptake blocker beta-alanine. While the basal release of [3H]GABA was not affected by CCK-8, the K(+)-stimulated release of [3H]GABA was significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [3H]GABA release was concentration-dependent and abolished by the CCKB receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCKA receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCKA and CCKB receptors are blocked. No CCK-8 effects on [3H]GABA release were observed when tetrodotoxin was superfused 5 min before the K(+)-induced [3H]GABA release. It is suggested that the enhancing actions of CCK-8 on K(+)-stimulated [3H]GABA release is mainly related to an activation of CCKB receptors.

Characteristics of pancreatic exocrine secretion produced by venom from the Brazilian scorpion, Tityus serrulatus.

The influence of venom (TSV) from the Brazilian scorpion, Tityus serrulatus, on exocrine pancreatic secretion was studied in relation to known cholinergic and peptidergic secretagogue activity. Pulse-labeling followed by chase incubation in the presence of secretagogues and various pharmacological agents revealed unique physiological characteristics of TSV in guinea pig pancreatic lobules. Exocytotic discharge of newly synthesized 3H-labeled proteins during a 3-h chase incubation showed a marked increase over basal discharge levels using logarithmic TSV doses of 0.10 to 100 micrograms/ml. This stimulation was comparable to maximal values elicited by carbachol, cholecystokinin-octapeptide (CCK-8) or caerulein and discharge kinetics were similar. TSV-mediated secretion was ATP and calcium dependent and partially inhibited by atropine. Only tetrodotoxin completely blocked TSV stimulation of newly synthesized protein discharge. Both botulinum toxin and curare had no effect on venom stimulation, indicating that TSV interaction with exocrine pancreatic cells occurs postsynaptically. Verapamil, a calcium channel antagonist, produced a moderate inhibition of TSV stimulation. When antagonists to the cholecystokinin (CCK) receptor were incubated with TSV, no change in secretory activity occurred. Therefore, TSV does not bind to CCK receptors and probably operates through its own receptor which may be an ion channel. Additionally, morphological studies in vitro revealed a high level of pancreatic secretory activity as evidenced by dense secretory acinar luminal content, reduction in zymogen granule (ZG) population, and development of exocytotic images.

Cholecystokinin-like immunoreactive retinal ganglion cells project to the ventral lateral geniculate nucleus in pigeons.

A subpopulation of retinal ganglion cells projecting to the pigeon ventral lateral geniculate nucleus was shown to contain cholecystokinin-like immunoreactivity. These ganglion cells were mainly distributed in the peripheral retina, and their somata sizes were medium to large (14-23 microns). Taken together with previous findings, these results indicate that the retinal input to the ventral geniculate is chemically heterogeneous.

[Biliary scintigraphy in patients with functional disorders of Oddi's sphincter]. / Cintigrafia biliar en pacientes con alteraciones funcionales del esfinter de Oddi.

The appearance of biliary pain after cholecystectomy in patients with normal ultrasonic and cholangiographic studies has been attributed to functional alterations of the Sphincter of Oddi. We performed dynamic cholescyntigraphic studies of the biliary tract in 32 cholecystectomized patients, at least six months after the operation: 27 were asymptomatic (control group) and 5 had clinical and laboratory findings suggesting temporary functional obstruction of the Sphincter of Oddi. In this group we demonstrated an emptying delay of the biliary tract that was modified by the IV infusion of Cholecystokinin Octapeptide. Biliary Cholescintigraphy appears as a good screening method to evaluate functional alterations of the Sphincter of Oddi.