Cholecystokinin and cholecystokinin-A receptor: An attractive treatment strategy for biliary dyskinesia?

Biliary dyskinesia is a relatively common gastrointestinal disease that is increas-ing in incidence as living standards improve.However,its underlying pathogenesis remains unclear,hindering the development of therapeutic drugs.Recently,“Expression and functional study of cholecystokinin-A receptors on the interstitial Cajal-like cells of the guinea pig common bile duct”demonstrated that cholecystokinin(CCK)regulates the contractile function of the common bile duct through interaction with the CCK-A receptor in interstitial Cajal-like cells,contributing to improving the academic understanding of biliary tract dynamics and providing emerging directions for the pathogenesis and clinical management of biliary dyskinesia.This letter provides a brief overview of the role of CCK and CCK-A receptors in biliary dyskinesia from the perspective of animal experiments and clinical studies,and discusses prospects and challenges for the clinical application of CCK and CCK-A receptors as potential therapeutic targets.

Serotonin receptor 2B induces visceral hyperalgesia in rat model and patients with diarrhea-predominant irritable bowel syndrome

BACKGROUND Serotonin receptor 2B(5-HT2B receptor)plays a critical role in many chronic pain conditions.The possible involvement of the 5-HT2B receptor in the altered gut sensation of irritable bowel syndrome with diarrhea(IBS-D)was investigated in the present study.AIM To investigate the possible involvement of 5-HT2B receptor in the altered gut sensation in rat model and patients with IBS-D.METHODS Rectosigmoid biopsies were collected from 18 patients with IBS-D and 10 patients with irritable bowel syndrome with constipation who fulfilled the Rome IV criteria and 15 healthy controls.The expression level of the 5-HT2B receptor in colon tissue was measured using an enzyme-linked immunosorbent assay and correlated with abdominal pain scores.The IBS-D rat model was induced by intracolonic instillation of acetic acid and wrap restraint.Alterations in visceral sensitivity and 5-HT2B receptor and transient receptor potential vanilloid type 1(TRPV1)expression were examined following 5-HT2B receptor antagonist adminis-tration.Changes in visceral sensitivity after administration of the TRPV1 antago-INTRODUCTION Irritable bowel syndrome(IBS)is a chronic functional bowel disorder characterized by recurrent abdominal pain with altered bowel habits that affects approximately 15%of the population worldwide[1].IBS significantly impacts the quality of life of patients.Although the pathogenesis of IBS is not completely understood,the role of abnormal visceral sensitivity in IBS has recently emerged[2,3].5-Hydroxytryptamine(5-HT)is known to play a key role in the physiological states of the gastrointestinal tract.Plasma 5-HT levels in IBS with diarrhea(IBS-D)patients were greater than those in healthy controls[4],suggesting a possible role of 5-HT in the pathogenesis of IBS-D.The serotonin receptor 2(5-HT2 receptor)family comprises three subtypes:5-HT2A,5-HT2B,and 5-HT2c.All 5-HT2 receptors exhibit 46%-50%overall sequence identity,and all of these receptors preferentially bind to Gq/11 to increase inositol phosphates and intracellular calcium mobilization[5].5-HT2B receptors are widely expressed throughout the gut,and experimental evidence suggests that the primary function of 5-HT2B receptors is to mediate contractile responses to 5-HT through its action on smooth muscle[6].The 5-HT2B receptor is localized to both neurons of the myenteric nerve plexus and smooth muscle in the human colon.The 5-HT2B receptor mediates 5-HT-evoked contraction of longitudinal smooth muscle[6].These findings suggest that the 5-HT2B receptor could play an important role in modulating colonic motility,which could affect sensory signaling in the gut.Other laboratories have shown that the 5-HT2B receptor participates in the development of mechanical and formalin-induced hyperalgesia[7,8].A 5-HT2B receptor antagonist reduced 2,4,6-trinitrobenzene sulfonic acid(TNBS)and stress-induced visceral hyperalgesia in rats[9,10].However,the role of the 5-HT2B receptor in IBS-D patients and in acetic acid-and wrap restraint-induced IBS-D rat models was not investigated.

Are TrkB receptor agonists the right tool to fulfill the promises for a therapeutic value of the brain-derived neurotrophic factor?

Brain-derived neurotrophic factor signaling via its receptor tro pomyosin receptor kinase B regulates several crucial physiological processes.It has been shown to act in the brain,promoting neuronal survival,growth,and plasticity as well as in the rest of the body where it is involved in regulating for instance aspects of the metabolism.Due to its crucial and very pleiotro pic activity,reduction of brain-derived neurotrophic factor levels and alterations in the brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling have been found to be associated with a wide spectrum of neurological diseases.Howeve r,because of its poor bioavailability and pharmacological properties,brain-derived neurotrophic factor itself has a very low therapeutic value.Moreover,the concomitant binding of exogenous brain-derived neurotrophic factor to the p75 neurotrophin receptor has the potential to elicit several unwanted and deleterious side effects.Therefo re,developing tools and approaches to specifically promote tropomyosin receptor kinase B signaling has become an important goal of translational research.Among the newly developed tools are different categories of tropomyosin receptor kinase B receptor agonist molecules.In this review,we give a comprehensive description of the diffe rent tro pomyosin receptor kinase B receptor agonist drugs developed so far and of the res ults of their application in animal models of several neurological diseases.Moreover,we discuss the main benefits of tropomyosin receptor kinase B receptor agonists,concentrating especially on the new tropomyosin receptor kinase B agonist antibodies.The benefits observed both in vitro and in vivo upon application of tropomyosin receptor kinase B receptor agonist drugs seem to predominantly depend on their general neuroprotective activity and their ability to promote neuronal plasticity.Moreover,tro pomyosin receptor kinase B agonist antibodies have been shown to specifically bind the tropomyosin receptor kinase B receptor and not p75 neurotrophin receptor.Therefore,while,based on the current knowledge,the tropomyosin receptor kinase B receptor agonists do not seem to have the potential to reve rse the disease pathology per se,promoting brainderived neurotrophic factor/tro pomyosin receptor kinase B signaling still has a very high therapeutic relevance.

Leukocyte immunoglobulin-like receptor B2:A promising biomarker for colorectal cancer

According to the latest global cancer statistics,colorectal cancer(CRC)has emerged as the third most prevalent malignant tumor across the globe.In recent decades,the medical field has implemented several levels of CRC screening tests,encompassing fecal tests,endoscopic examinations,radiological examinations and blood tests.Previous studies have shown that leukocyte immunoglobulin-like receptor B2(LILRB2)is involved in inhibiting immune cell function,immune evasion,and promoting tumor progression in acute myeloid leukemia and nonsmall cell lung cancer.However,its interaction with CRC has not been reported yet.Recently,a study published in the World Journal of Gastroenterology revealed that LILRB2 and its ligand,angiopoietin-like protein 2,are markedly overexpressed in CRC.This overexpression is closely linked to tumor progression and is indicative of a poor prognosis.The study highlights the potential of utilizing the concentration of LILRB2 in serum as a promising biomarker for tumors.However,there is still room for discussion regarding the data processing and analysis in this research.

Leukocyte immunoglobulin-like receptor B2 overexpression as a promising therapeutic target and noninvasive screening biomarker for colorectal cancer

BACKGROUND Colorectal cancer(CRC)has become the second most deadly malignancy in the world,and the exploration of screening markers and precise therapeutic targets is urgent.Our previous research identified leukocyte immunoglobulin-like receptor B2(LILRB2)protein as a characteristic protein of CRC,but the association between LILRB2 expression and clinicopathological features,the internal mechanism related to CRC progression,and screening diagnostic efficacy are not clear.Therefore,we hypothesized that LILRB2 is significantly highly expressed in CRC tissues,correlated with advanced stage and a poor prognosis,and could be used as a therapeutic target and potential screening biomarker for CRC.AIM To explore whether LILRB2 can be used as a potential therapeutic target and noninvasive screening biomarker for CRC.METHODS Patients who underwent radical surgery for CRC at China-Japan Friendship Hospital between February 2021 and October 2022 were included.Cancer and paracancerous tissues were collected to verify LILRB2 expression,and the association between LILRB2 expression and clinicopathological features was analysed.Serum was collected from CRC patients,adenoma patients and healthy controls during the same period to assess the diagnostic value of LILRB2 as a noninvasive screening biomarker,and its diagnostic value was further compared with that of the traditional markers carcinoembryonic antigen(CEA)and carbohydrate antigen 19-9(CA19-9).RESULTS A total of 58 CRC patients were included,and LILRB2 protein was significantly overexpressed in cancer tissues compared with paracancerous tissues(P<0.001).Angiopoietin-like protein 2(ANGPTL2)protein,as the ligand of LILRB2,was synergistically overexpressed in CRC tissues(P<0.001),and overexpression of LILRB2 and ANGPTL2 protein was significantly correlated with poor to moderate differentiation,vascular involvement,lymph node metastasis,distant metastasis,advanced tumor-node-metastasis stage and a poor prognosis(P<0.05),which suggested that LILRB2 and ANGPTL2 are closely associated with CRC progression.In addition,serum LILRB2 concentrations increased stepwise in healthy individuals,adenoma patients and CRC patients with statistically significant differences.The sensitivity of serum LILRB2 for the diagnosis of CRC was 89.74%,the specificity was 88.89%,the area under the curve was 0.95,and the diagnostic efficacy was better than that of conventional CEA and CA19-9.CONCLUSION LILRB2 protein can be used as a potential novel therapeutic target and noninvasive screening biomarker for CRC,which is beneficial for early screening and precise treatment.

Association of vitamin D and polymorphisms of its receptor with antiviral therapy in pregnant women with hepatitis B

BACKGROUND The interruption of mother-to-child transmission(MTCT)is considered important to decrease the individual and population morbidity of hepatitis B virus(HBV)infection as well as the global burden of hepatitis B.Serum vitamin D(VD)is associated with hepatitis B.AIM To assess whether baseline VD levels and single nucleotide polymorphisms of the VD receptor gene(VDR SNPs)are associated with the efficacy of tenofovir disoproxil fumarate(TDF)in the prevention of MTCT in pregnant women with high HBV viral loads.METHODS Thirty-eight pregnant women who were at high risk for MTCT of HBV(those with an HBV DNA level≥2×10^(5)IU/mL during 12-24 wk of gestation)receiving antiviral therapy of TDF between June 1,2019 and June 30,2021 in Mianyang were included in this retrospective study.The women received 300 mg TDF once daily from gestational weeks 24-28 until 3 mo after delivery.To further characterize the clinical relevance of maternal serum HBV DNA levels,we stratified patients according to HBV DNA level as follows:Those with levels<2×10_(5)(full responder group)vs those levels≥2×10^(5)IU/mL(partial responder group)at delivery.Serum levels of 25-hydroxyvitamin D[25(OH)D],liver function markers,virological parameters,VDR SNPs and other clinical parameters were collected to analyze their association with the efficacy of TDF.The Mann-Whitney U test or t test was used to analyze the serum levels of 25(OH)D in different groups.Multiple linear regressions were utilized to analyze the determinants of the maternal HBV DNA level at delivery.Univariate and multivariate logistic regression analyses were employed to explore the association of targeted antiviral effects with various characteristics at baseline and delivery.RESULTS A total of 38 pregnant women in Mianyang City at high risk for MTCT of HBV were enrolled in the study.The MTCT rate was 0%.No mother achieved hepatitis B e antigen or hepatitis B surface antigen(HBsAg)clearance at delivery.Twenty-three(60.5%)participants were full responders,and 15(39.5%)participants were partial responders according to antiviral efficacy.The present study showed that a high percentage(76.3%)of pregnant women with high HBV viral loads had deficient(<20 ng/mL)or insufficient(≥20 but<31 ng/mL)VD levels.Serum 25(OH)D levels in partial responders appeared to be significantly lower than those in full responders both at baseline(25.44±9.42 vs 17.66±5.34 ng/mL,P=0.006)and delivery(26.76±8.59 vs 21.24±6.88 ng/mL,P=0.044).Serum 25(OH)D levels were negatively correlated with maternal HBV DNA levels[log(10)IU/mL]at delivery after TDF therapy(r=-0.345,P=0.034).In a multiple linear regression analysis,maternal HBV DNA levels were associated with baseline maternal serum 25(OH)D levels(P<0.0001,β=-0.446),BMI(P=0.03,β=-0.245),baseline maternal log10 HBsAg levels(P=0.05,β=0.285)and cholesterol levels at delivery(P=0.015,β=0.341).Multivariate logistic regression analysis showed that baseline serum 25(OH)D levels(OR=1.23,95%CI:1.04-1.44),maternal VDR Cdx2 TT(OR=0.09,95%CI:0.01-0.88)and cholesterol levels at delivery(OR=0.39,95%CI:0.17-0.87)were associated with targeted antiviral effects(maternal HBV DNA levels<2×10^(5) at delivery).CONCLUSION Maternal VD levels and VDR SNPs may be associated with the efficacy of antiviral therapy in pregnant women with high HBV viral loads.Future studies to evaluate the therapeutic value of VD and its analogs in reducing the MTCT of HBV may be justified.

Expression profile of cholecystokinin type-A receptor in gallbladder cancer and gallstone disease

BACKGROUND:Regulatory peptide receptors have attracted the interest of oncologists as a new promising approach for cancer pathology,imaging and therapy.Although cholecystokinin (CCK) is a potent modulator of gallbladder contractility and plays a potential role in pancreatic carcinogenesis through CCK type-A receptor (CCKAR),its role in gallbladder cancer (GBC) is still unknown and immunohistochemical detection of CCKAR in the gallbladder has not yet been reported.This novel case-control study aimed to investigate the expression profile of CCKAR in GBC and gallstone disease (GSD).METHODS:This study included 162 samples of gallbladder:94 from GBC and 68 from GSD.Expression of CCKAR was analyzed by immunohistochemistry and immunoblotting.The results were statistically correlated with disease history including age,sex,presence of gallstone,stage and differentiation.RESULTS:CCKAR was positive in 30/68 (44.1%) of GSD and 72/94 (76.6%) of GBC samples.Fifty-one of the 72 (70.8%) CCKAR-positive GBC samples showed over-expression.Interestingly,consistent results also appeared in the immunoblotting study.CONCLUSIONS:CCKAR expression was significantly increased in GBC compared to GSD.Moreover,CCKAR expression was associated with the degree of tumor differentiation,i.e.,less expression in poorly-differentiated tumors.Thus,it has future prognostic and therapeutic implications in the management of GBC.

Expression of gastrin and cholecystokinin B receptor in Lateolabrax maculatus

Gastrin(gas)is a peptide hormone that stimulates gastric acid secretion by gastric parietal cells and stimulates gastric motility.The cholecystokinin B receptor(cckbr)can act as a receptor for gastrin,conveying regulatory information on gastrin,but there are fewer studies on its function in fish.The Lateolabrax maculatus is one of the marine aquaculture species in China,it widely distribute in coastal areas.In the study,we cloned the genes of Lateolabrax maculatus gastrin(Lm-gas)and Lateolabrax maculatus cholecystokinin B receptor(Lm-cckbr).The results showed that the full-length gene of Lm-gas is 638bp and the carboxy-terminal conserved domain(DFGRR)is the core functional domain of gastrin protein.The Lm-cckbr gene has a total nucleotide sequence of 2066 bp,and the open reading frame encodes a total of 453 amino acids.The result of protein sequence alignment showed that the similarity between Lm-cckbr protein and other different species was 50.11%-89.67%.The PCR results showed that Lm-gas and Lm-cckbr were expressed in brain and stomach.Further localization by immunehistochemical staining showed that Lm-gas protein was located in the mucosal layer of the gastric wall,but the expression signal was weak in the brain.Hunger causeed a significant decrease in these two genes.The results provided basic research data for further study on the function of Lm-gas and its recepter Lm-cckbr in the in the central nervous system and digestive system of Lateolabrax maculatus.

Gallbladder motor function, plasma cholecystokinin and cholecystokinin receptor of gallbladder in cholesterol stone patients

AIM: To study the interactive relationship of gallbladder motor function, plasma cholecystokinin (CCK) and cholecystokinin A receptor (CCK-R) of gallbladder in patients with cholesterol stone disease.METHODS: Gallbladder motility was studied by ultrasonography in 33 patients with gallbladder stone and 10 health subjects as controls. Plasma CCK concentration was measured by radioimmunoassay in fasting status (CCK-f) and in 30 min after lipid test meal (CCK-30).Radioligand method was employed to analyze the amount and activity of CCK-R from 33 gallstone patients having cholecystectomy and 8 persons without gallstone died of severe trauma as controls.RESULTS: The percentage of cholesterol in the gallstone composition was more than 70%. The cholesterol stone type was indicated for the patients with gallbladder stone in this study. Based on the criterion of gallbladder residual fraction of the control group, 33 gallstone patients were divided into two subgroups, contractor group (14 cases)and non-contractor group (19 cases), The concentration of CCK-30 was significantly higher in non-contractor group than that in both contractor group and control group (55.86±3.86 pmol/l vs 37.85±0.88 pmol/l and 37.95±0.74 pmol/L, P＜0.01), but there was no difference between contractor group and control group. Meanwhile no significant difference of the concentration of CCK-f could be observed among three groups. The amount of CCK-R was lower in non-contractor group than those in both control group and contractor group (10.27±0.94 fmol/mg vs24.59±2.39 fmol/mg and 22.66±0.55 fmol/mg, P＜0.01).The activity of CCK-R shown as KD in non-contractor group decreased compared to that in control group and contractor group. Only was the activity of CCK-R lower in contractor group than that in control group. The ejection fraction correlated closely with the amount of CCK-R (r = 0.9683,P＜0.01), and the concentration of CCK-30 correlated negatively with the amount of CCK-R closely (r = -0.9627,P＜0.01).CONCLUSION: The distinctive interactive relationship of gallbladder emptying, plasma CCK and CCK-R in gallbladder from this study suggested that the defect of CCK-R may be a key point leading to the impairment of gallbladder motor function and the pathogenesis of cholesterol gallstoneformation may differ in two subgroups of gallstone patient,gallbladder non-contractor group or contractor group.

Effects of gamma-aminobutyric acid receptors on muscarinic receptor-mediated free calcium ion levels in the facial nucleus following facial nerve injury

Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A （GABAA） receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B （GABAB） and C （GABAc） receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.

Control of gallbladder contractions by cholecystokinin through cholecystokinin-A receptors on gallbladder interstitial cells of cajal

AIM: To identify the cholecystokinin (CCK)-A receptors (CCK-AR) on the guniea pig gallbladder interstitial cells of cajal (ICC) and to study CCK-8 induced gallbladder muscle strip contractions through the CCK-AR. METHODS: The existence of CCK-AR was examined by immunohistofluorescence on sectioned tissue and cultured cells. In vitro contractile response of guinea pig gallbladder muscle strips and the strips with ICC removed were also studied with CCK-8 receptors added. RESULTS: In tissue sections, intensely CCKAR- immunoreactive interstitial cells were found mainly in the muscular layers. In cultured cell sections, distinctive double staining of C-kit and CCK-AR ICCs were found. When we removed the ICC of the gallbladder, CCK-8 induced muscle strip contraction dose response curve significantly shifted to the right. CONCLUSION: We proved that both the existence of CCK-AR on the guinea pig gallbladder ICC and CCK evoked contraction are mediated through direct action on CCK-AR on the gallbladder ICC.

Blockade of cholecystokinin-2 receptor and cyclooxygenase-2 synergistically induces cell apoptosis, and inhibits the proliferation of human gastric cancer cells in vitro

Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer.Here,we demonstrated that the combination of AG-041R(a CCK-2 receptor antagonist) plus NS-398(a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition,apoptosis induction,down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells.These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.(C)2008 Elsevier Ireland Ltd.All rights reserved.

Correlation between Pulmonary Endothelin Receptors and Alveolar-arterial Oxygen Gradient in Rats with Hepatopulmonary Syndrome

The correlation between pulmonary endothelin receptors and alveolar-arterial oxygen gradient （A aDO2） in rats with hepatopulmonary syndrome was investigated. Animals were divided into 2 groups： Sham operated （Sham） group and common bile duct ligation （CBDL） group. Arterial blood gas was evaluated by a blood gas analyzer. The concentrations of ET-1 in blood and lung tissue sample were evaluated by radioimmunoassay. The distribution and expression of two kinds of subtype receptor of ET-1, ETRA and ETRB were examined by in situ hybridization. The results showed that the level of A aDO2, was higher in CBDI. group than that in Sham group （P〈0.05）. The levels of plasma and pulmonary ET-1 in CBDL group were both higher than in Sham group （P〈0.05 ）. There was no significant difference in average A of ETRA between two groups by imaging analysis （0.21±0.06 vs 0.22±0.08, P〉0.05）, while that of ETRB was higher in CBDI. group than in Sham group （0.58±0.16 vs 0.28±0.07, P〈0.05）. The expression of ETRBinlung was positively correlated with A aDO2（P〈0.05）. It was concluded that the widened A-aDO2 may be related with enhancement of the expression of ETRB in lung.

Expression of ΔDNMT3B Variants and Its Association with Estrogen/Progestogen Receptor Status in Breast Cancer

Objective： Our previous study has showed that △DNMT3B is the predominant form of DNMT3B in non-small cell lung cancer （NSCLC）. In this study, we aimed to explore the expression patterns of the △DNMT3B variants in breast cancer and to identify whether the pattern was similar to that in NSCLC or not and its clinical significance. Methods： Expression of seven △DNMT3B variants in 59 breast cancer and the corresponding normal tissue was measured using RT-PCR. The correlations between the expressions of △DNMT3B variants and the clinical parameters including ER/PR status, clincopathologic feature and survivals were analyzed. Results： There were significant differences in the expression ratios of △DNMT3B1-7 variants between breast cancer tissues and normal tissues （P〈0.001）. The positive ratio of △DNMT3B1-7 variants were 66%, 71%, 17%, 51%, 76.2%, 50% and 61% in tumor tissue, respectively; while 16%, 8.4%, 3.38%, 3.38%, 11.8%, 13.5% and 5.08% in the corresponding normal tissue, which was different from the pattern of △DNMT3B1-7 expression in NSCLC （62%, 76%, 2.5%, 46%, 18%, 27% and 16% in tumor tissue, respectively; while 18%, 11%, 0%, 3.3%, 0%, 0% and 0% in normal lung tissue, respectively; P〈0.0001）. Expressions of △DNMT3B2, 3B4 and 3B7 were higher in the patients with negative estrogen receptor （ER） than those with positive estrogen receptor （P=0.035, P=0.0141 and P=0.0219, respectively）. △DNMT3B7 expression was higher for the patients with negative progestogen receptor （PR） compared to those with positive progestogen receptor （P=0.0379）. Expression ratio of △DNMT3B5 in stage Ⅲ tumors is lower than that in stage Ⅰ/Ⅱ ones （P= 0.041）. But we did not find any relation between the △DNMT3B variants and the patients＇ survival. Conclusion： The pattern of △DNMT3B variants in breast cancer is different from that in NSCLC. Expressions of △DNMT3B2, 3B4 and 3B7 are associated with estrogen receptors status. While △DNMT3B7 is associated with progestogen receptor. No relation between the △DNMT3B variants and the patients＇ survival were found.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.

Investigation of cholecystokinin receptors in the human lower esophageal sphincter

AIM:To compare the binding of cholecystokinin(CCK)-8to CCK receptors in sling and clasp fibers of the human lower esophageal sphincter.METHODS:Esophageal sling and clasp fibers were isolated from eight esophagectomy specimens,resected for squamous cell carcinoma in the upper two thirds of the esophagus,which had been maintained in oxygenated Kreb’s solution.Western blot was used to measure CCK-A and CCK-B receptor subtypes in the two muscles.A radioligand binding assay was used to determine the binding parameters of 3H-CCK-8S to the CCK receptor subtypes.The specificity of binding was determined by the addition of proglumide,which blocks the binding of CCK to both receptor subtypes.RESULTS:There was no significant difference between the sling and clasp fibers of the human lower esophageal sphincter in the amount of CCK-A[integratedoptical density(IOD)value:22.65±0.642 vs 22.328±1.042,P=0.806]or CCK-B receptor protein(IOD value:13.20±0.423 vs 12.45±0.294,P=0.224)as measured by Western blot.The maximum binding of radio-labeled CCK-8S was higher in the sling fibers than in the clasp fibers(595.75±3.231 cpm vs 500.000±10.087 cpm,P<0.001)and dissociation constant was lower(Kd:1.437±0.024 nmol/L vs 1.671±0.024nmol/L,P<0.001).The IC50 of the receptor specific antagonists were lower for the CCK-A receptors than for the CCK-B(P<0.01).CONCLUSION:CCK binding modulates the contractile function of the lower esophageal sphincter through differential binding to the CCK-A receptor on the sling and clasp fibers.

Increased endothelin receptor B and G protein coupled kinase-2 in the mesentery of portal hypertensive rats

AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.

Scavenger receptor BI: A multi-purpose player in cholesterol and steroid metabolism

Scavenger receptor class B type Ⅰ (SR-BI) is an important member of the scavenger receptor family of integral membrane glycoproteins. This review highlights studies in SR-BI knockout mice, which concern the role of SR-BI in cholesterol and steroid metabolism. SR-BI in hepatocytes is the sole molecule involved in selective uptake of cholesteryl esters from high-density lipoprotein (HDL). SR-BI plays a physiological role in binding and uptake of native apolipoprotein B (apoB)-containing lipoproteins by hepatocytes, which identif ies SR-BI as a multipurpose player in lipid uptake from the blood circulation into hepatocytes in mice. In adrenocortical cells, SR-BI mediates the selective uptake of HDL-cholesteryl esters, which is eff iciently coupled to the synthesis of glucocorticoids (i.e. corticosterone). SR-BI knockout mice suffer from adrenal glucocorticoid insuff iciency, which suggests that functional SR-BI protein is necessary for optimal adrenal steroidogenesis in mice. SR-BI in macrophages plays a dual role in cholesterol metabolism as it is able to take up cholesterol associated with HDL and apoBcontaining lipoproteins and can possibly facilitate cholesterol efflux to HDL. Absence of SR-BI is associated with thrombocytopenia and altered thrombosis susceptibility, which suggests a novel role for SR-BI in regulating platelet number and function in mice. Transgenic expression of cholesteryl ester transfer protein in humanized SR-BI knockout mice normalizes hepatic delivery of HDL-cholesteryl esters. However, other pathologies associated with SR-BI def iciency, i.e. increased atherosclerosis susceptibility, adrenal glucocorticoid insuffi ciency, and impaired platelet function are not normalized, which suggests an important role for SR-BI in cholesterol and steroid metabolism in man. In conclusion, generation of SR-BI knockout mice has signif icantly contributed to our knowledge of the physiological role of SR-BI. Studies using these mice have identif ied SR-BI as a multi-purpose player in cholesterol and steroid metabolism because it has distinct roles in reverse cholesterol transport, adrenal steroidogenesis, and platelet function.

Upregulation of Toll-like Receptor 4 on T Cells in PBMCs Is Associated with Disease Aggravation of HBV-related Acute-on-chronic Liver Failure

Summary： Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure （ACLF）. Toll-like receptors （TLRs） have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was.to characterize the TLR4 expression in peripheral blood mononuclear cells （PBMCs） of ACLF pa- tients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected （CHB） patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 ex- pression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was ana- lyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expres- sion of TLR4 on CD4＋ and CD8＋ T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4＋ and CD8＋T cells were positively correlated with serum total bilirubin （TBIL）, direct bilirubin （DBIL）, international normalized ratio （INR） levels and white blood cells （WBCs）, and negatively correlated with serum albumin （ALB） levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4＋ T cells, which was also posi- tively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.