ObjectiveTo investigate the effects of Yueju Wan on the 5-hydroxytryptamine （5-HT） signaling pathway in rats with functional dyspepsia （FD） and to explore its therapeutic mechanism in the treatment of FD. MethodsSixty Sprague-Dawley （SD） rats were randomly divided into a normal group， model group， mosapride group （1.575 mg·kg^-1）， and Yueju Wan low-， medium-， and high-dose groups （0.735， 1.47， and 2.94 g·kg^-1， respectively）. The FD rat model was established using GUO's tail-clamping stimulation combined with irregular feeding. After 14 days of modeling， rats were administered the corresponding drugs by gavage for 28 days. After treatment， gastric emptying rate and small intestinal propulsion rate were measured. Serum levels of 5-HT， tryptophan hydroxylase （TPH）， and substance P （SP） were detected by enzyme-linked immunosorbent assay （ELISA）， and acetylcholine （ACh） levels were determined by chemical methods. Histopathological changes in the gastric antrum were observed using hematoxylin-eosin （HE） staining. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to assess the mRNA and protein expression levels of 5-hydroxytryptamine 4 receptor （5-HT4R）， SP， and acetylcholinesterase （AChE） in colon tissue， as well as 5-hydroxytryptamine 3 receptor （5-HT3R）， SP， and AChE in hypothalamic tissue. Immunohistochemistry （IHC） was used to examine the expression of 5-HT and 5-HT4R in the colon and 5-HT and 5-HT3R in the hypothalamus. ResultsCompared with the normal group， the gastric emptying rate and small intestinal propulsion rate in the model group were significantly decreased （P<0.01）. Serum levels of 5-HT， SP， ACh， and TPH were significantly reduced （P<0.01）. Histopathological examination revealed irregular arrangement of glands in the gastric antrum， slight mucosal atrophy， and mild inflammatory cell infiltration. The mRNA and protein expression levels of 5-HT4R， SP， and AChE in colon tissue， as well as 5-HT3R， SP， and AChE in hypothalamic tissue， were significantly decreased （P<0.01）， and 5-HT protein expression in both the colon and hypothalamus was also significantly reduced （P<0.01）. Compared with the model group， all Yueju Wan groups showed significantly increased gastric emptying rate and small intestinal propulsion rate （P<0.01）. The glands in the gastric antrum were more regularly arranged， with no inflammatory cell infiltration observed. Serum levels of 5-HT， SP， ACh， and TPH were significantly increased （P<0.01）. The mRNA and protein expression levels of 5-HT4R， SP， and AChE in colon tissue and 5-HT3R， SP， and AChE in hypothalamic tissue were significantly upregulated （P<0.05， P<0.01）， and 5-HT protein expression in both the colon and hypothalamus was significantly increased （P<0.01）. ConclusionYueju Wan has preventive and therapeutic effects on FD， and its mechanism may be related to regulation of the 5-HT signaling pathway， promotion of brain-gut peptide secretion， and enhancement of gastric motility.

ObjectiveTo investigate the effect of bone morphogenetic protein 3 （BMP3） on the expression of inflammatory factors and joint damage in adjuvant arthritis （AA） induced by Freund′s complete adjuvant （FCA） in rats. MethodsThe AIA model was established in SD rats by intradermal injection of FCA into the toes of the left hind limb， and BMP3 overexpressing adenovirus （Ad-BMP3） or control adenovirus （Ad-NC） was injected in situ into the knee joint cavity on day 8 after modeling. Subsequently， HE staining was used to observe the histopathological changes in the synovium， immunohistochemistry was used to detect the expression of BMP3 in the synovium， and ELISA was used to analyze the expression levels of IL-6， IL-1β and TNF-α in the serum. Primary fibroblast-like synoviocytes （FLS） were isolated from AIA rats， the expression of BMP3 in FLS was knocked down or overexpressed， and Western blot and qRT-PCR were used to detect the expression levels of BMP3 and inflammatory factors in FLS. ResultsHE staining confirmed the successful establishment of the AIA model. Compared with normal rats， AIA rats showed decreased BMP3 expression in synovial tissue. Knockdown of BMP3 promoted the protein expression of inflammatory factors （IL-6， IL-1β， IL-17A， TNF-α） and the mRNA expression of chemokines ［C-C motif chemokine ligand 2 （CCL2）， C-C motif chemokine ligand 3 （CCL3）， Vascular Cell Adhesion Molecule-1 （VCAM-1）］ in FLS. In contrast， overexpression of BMP3 suppressed the expression of these inflammatory factors and chemokines. Intra-articular injection of BMP3-overexpressing adenovirus in AIA rats upregulated BMP3 expression in synovial tissue and inhibited synovial inflammation and bone erosion. ConclusionBMP3 suppresses the production of inflammatory factors and chemokines in FLS， thereby alleviating synovial hyperplasia and bone erosion in arthritis.

Objective:To verify and compare the correlation and diagnostic efficacy of international bowel ultrasound segmental activity score（IBUS-SAS），bowel ultrasound score（BUSS），simple ultrasound score for Crohn's disease（SUS-CD），and simple ultrasound score（Simple-US）with endoscopic disease activity in Crohn's disease（CD）patients. To provide external validation of the diagnostic efficacy of intestinal ultrasound（IUS）score and theoretical basis for clinical selection of optimal IUS score.Methods:A total of 160 patients with clinical diagnosis of CD combined with IUS and intestinal endoscopy were retrospectively analyzed in the Second Affiliated Hospital of Zhejiang University School of Medicine from January 2022 to August 2024. IUS parameters were measured and scored with IBUS-SAS，SUS-CD，BUSS and Simple-US scores. Endoscopic SES-CD was used to evaluate intestinal disease activity in patients without history of intestinal resection，and Rutgeerts score was used to evaluate intestinal disease activity in patients with history of intestinal resection. Endoscopic remission in patients with CD was defined as SES-CD < 3 or Rutgeerts score i0 and i1，mild endoscopic disease activity was defined as 7 > SES-CD≥3 or Rutgeerts score = 2，moderate endoscopic disease activity was defined as 15 > SES-CD≥7 or Rutgeerts score i3，severe endoscopic disease activity was defined as SES-CD≥15 or Rutgeerts i4. The correlation and diagnostic efficacy of IBUS-SAS，SUS-CD，BUSS and Simple-US scores with endoscopic disease activity in patients with CD were compared and analyzed.Results:IUS scores including IBUS-SAS，SUS-CD，BUSS and Simple-US were significantly correlated with endoscopic intestinal disease activity，SES-CD and Rutgeerts scores in CD patients（ r s = 0.706，0.492，0.502，0.526；0.825，0.581，0.584，0.603；0.541，0.434，0.437，0.467；all P＜0.05）. Among them，IBUS-SAS showed better correlation than the other three IUS scores. ROC curve showed that IBUS-SAS，SUS-CD，BUSS，and Simple-US had high predictive values for endoscopic disease activity and endoscopic disease moderate-severe activity in patients with CD（AUC = 0.886，0.748，0.730，0.756；all P＜0.05）. The diagnostic efficacy of IBUS-SAS on the presence of endoscopic disease activity in patients with CD was significantly higher than those of the other three IUS scores . Conclusions:IBUS-SAS，SUS-CD，BUSS and Simple-US are significantly correlated with the endoscopic results of on intestinal disease activity of CD，and have high predictive values for intestinal disease activity status，among which IBUS-SAS is superior to the other three IUS scores. It is recommended that IBUS-SAS be used first to evaluate intestinal disease activity in patients with CD.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

Objective:To analyze the effect of mouse nerve growth factor(mNGF)combined with rehabilitation training on the expression of BDNF and Caspase-3 in brain tissue of newborn rats with spastic cerebral palsy(SCP).Method:A total of 150 clean-grade SD rats aged 7days were randomly divided into the control group(group NS,n=40)and the model group(group T,n=110)using a random number table.Group T received directional injection of absolute ethanol(1μl/g)into the left intracranial pyramidal tract at the age of 7 days,while the NS group received an equal volume of saline.24 hours after injection(at 8 days old),the spastic cerebral palsy rat model was determined by modified Li Xiaojie et al.'s criteria combined with Longa scoring method.Ninety successfully modeled SD rats in group T were randomly divided into group A1,A2 and A3,with 30 each.The intervention began on the 1st day of the experiment(8 days old).Group A1 was given intramuscu-lar injection of mNGF(30ng/g),group A2 was given intramuscular injection of an equal dose of mNGF plus rehabilitation training,and group A3 was given intramuscular injection of an equal volume of saline(2.27ml/kg).The group NS was given intramuscular injection of the equal volume of normal saline.Intervention lasted until 21 days of age.At 8,14 and 21 days of age,HE staining was used to observe the dynamic changes in the morphology of cells in the internal capsule area of brain,TUNEL staining to observe the dynamic changes in the apoptosis rate of cells in the internal capsule area of brain,and Western Blot to monitor dy-namic changes in the expression of BDNF and Caspase-3 proteins in the hippocampus.Result:Model preparation showed that a total of 97 spastic cerebral palsy litters were identified in group T at 8 days old,while no rats with cerebral palsy in groupNS.TUNELstaining revealed that the apoptosis rates of the rats in group A1,A2 and A3 were significantly higher than in group NS at each age(P<0.01),and were significantly higher in group A3 than in group A1 and A2 at 14 and 21 days of age(P<0.01),with group A1 significantly higher than group A2(P<0.01).Western Blot showed that the relative expression of BDNF and Caspase-3 protein in group A1,A2 and A3 at 14 days old were higher than that in group NS(P<0.05).The rel-ative expression of BDNF protein in group A1 and A2 was higher than that in group A3(P<0.05),with group A2 was higher than that in group A1(P<0.05).There was no significant difference between group A2 and A1 at 21 days old(P>0.05).The relative expression of Caspase-3 protein in group A1 and A3 was higher than that in group A2(P<0.05).and group A3 was significantly higher than that in A1 group(P<0.05).Conclusion:It is speculated that both mNGF and combined rehabilitation training can inhibit neuronal apoptosis by promoting BDNF protein expression and reducing the activity of Caspase-3.The effect of mNGF combined with rehabilitation training is better than that of mNGF alone.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

Objective:To analyze the effect of mouse nerve growth factor(mNGF)combined with rehabilitation training on the expression of BDNF and Caspase-3 in brain tissue of newborn rats with spastic cerebral palsy(SCP).Method:A total of 150 clean-grade SD rats aged 7days were randomly divided into the control group(group NS,n=40)and the model group(group T,n=110)using a random number table.Group T received directional injection of absolute ethanol(1μl/g)into the left intracranial pyramidal tract at the age of 7 days,while the NS group received an equal volume of saline.24 hours after injection(at 8 days old),the spastic cerebral palsy rat model was determined by modified Li Xiaojie et al.'s criteria combined with Longa scoring method.Ninety successfully modeled SD rats in group T were randomly divided into group A1,A2 and A3,with 30 each.The intervention began on the 1st day of the experiment(8 days old).Group A1 was given intramuscu-lar injection of mNGF(30ng/g),group A2 was given intramuscular injection of an equal dose of mNGF plus rehabilitation training,and group A3 was given intramuscular injection of an equal volume of saline(2.27ml/kg).The group NS was given intramuscular injection of the equal volume of normal saline.Intervention lasted until 21 days of age.At 8,14 and 21 days of age,HE staining was used to observe the dynamic changes in the morphology of cells in the internal capsule area of brain,TUNEL staining to observe the dynamic changes in the apoptosis rate of cells in the internal capsule area of brain,and Western Blot to monitor dy-namic changes in the expression of BDNF and Caspase-3 proteins in the hippocampus.Result:Model preparation showed that a total of 97 spastic cerebral palsy litters were identified in group T at 8 days old,while no rats with cerebral palsy in groupNS.TUNELstaining revealed that the apoptosis rates of the rats in group A1,A2 and A3 were significantly higher than in group NS at each age(P<0.01),and were significantly higher in group A3 than in group A1 and A2 at 14 and 21 days of age(P<0.01),with group A1 significantly higher than group A2(P<0.01).Western Blot showed that the relative expression of BDNF and Caspase-3 protein in group A1,A2 and A3 at 14 days old were higher than that in group NS(P<0.05).The rel-ative expression of BDNF protein in group A1 and A2 was higher than that in group A3(P<0.05),with group A2 was higher than that in group A1(P<0.05).There was no significant difference between group A2 and A1 at 21 days old(P>0.05).The relative expression of Caspase-3 protein in group A1 and A3 was higher than that in group A2(P<0.05).and group A3 was significantly higher than that in A1 group(P<0.05).Conclusion:It is speculated that both mNGF and combined rehabilitation training can inhibit neuronal apoptosis by promoting BDNF protein expression and reducing the activity of Caspase-3.The effect of mNGF combined with rehabilitation training is better than that of mNGF alone.

Objective:To verify and compare the correlation and diagnostic efficacy of international bowel ultrasound segmental activity score（IBUS-SAS），bowel ultrasound score（BUSS），simple ultrasound score for Crohn's disease（SUS-CD），and simple ultrasound score（Simple-US）with endoscopic disease activity in Crohn's disease（CD）patients. To provide external validation of the diagnostic efficacy of intestinal ultrasound（IUS）score and theoretical basis for clinical selection of optimal IUS score.Methods:A total of 160 patients with clinical diagnosis of CD combined with IUS and intestinal endoscopy were retrospectively analyzed in the Second Affiliated Hospital of Zhejiang University School of Medicine from January 2022 to August 2024. IUS parameters were measured and scored with IBUS-SAS，SUS-CD，BUSS and Simple-US scores. Endoscopic SES-CD was used to evaluate intestinal disease activity in patients without history of intestinal resection，and Rutgeerts score was used to evaluate intestinal disease activity in patients with history of intestinal resection. Endoscopic remission in patients with CD was defined as SES-CD < 3 or Rutgeerts score i0 and i1，mild endoscopic disease activity was defined as 7 > SES-CD≥3 or Rutgeerts score = 2，moderate endoscopic disease activity was defined as 15 > SES-CD≥7 or Rutgeerts score i3，severe endoscopic disease activity was defined as SES-CD≥15 or Rutgeerts i4. The correlation and diagnostic efficacy of IBUS-SAS，SUS-CD，BUSS and Simple-US scores with endoscopic disease activity in patients with CD were compared and analyzed.Results:IUS scores including IBUS-SAS，SUS-CD，BUSS and Simple-US were significantly correlated with endoscopic intestinal disease activity，SES-CD and Rutgeerts scores in CD patients（ r s = 0.706，0.492，0.502，0.526；0.825，0.581，0.584，0.603；0.541，0.434，0.437，0.467；all P＜0.05）. Among them，IBUS-SAS showed better correlation than the other three IUS scores. ROC curve showed that IBUS-SAS，SUS-CD，BUSS，and Simple-US had high predictive values for endoscopic disease activity and endoscopic disease moderate-severe activity in patients with CD（AUC = 0.886，0.748，0.730，0.756；all P＜0.05）. The diagnostic efficacy of IBUS-SAS on the presence of endoscopic disease activity in patients with CD was significantly higher than those of the other three IUS scores . Conclusions:IBUS-SAS，SUS-CD，BUSS and Simple-US are significantly correlated with the endoscopic results of on intestinal disease activity of CD，and have high predictive values for intestinal disease activity status，among which IBUS-SAS is superior to the other three IUS scores. It is recommended that IBUS-SAS be used first to evaluate intestinal disease activity in patients with CD.

Objective To investigate the therapeutic effect of isoorientin on ulcerative colitis(UC)mice induced by dextran sulfate sodium(DSS)and its mechanism.Methods Forty-eight C57BL/6J mice were randomly divided into control group,model group,mesalazine group(600 mg·kg-1),isoorientin low dose group(25 mg·kg-1),isoorientin high dose group(50 mg·kg-1)and isoorientin control group(50 mg·kg-1),with eight mice in each group.Mice were free to drink 2%DSS solution to replicate UC model.After three weeks of experiment,each drug administration group was given corresponding drug by intragastric administration for four weeks.After the last administration,the body weight was weighed,blood was taken from eyeballs,and colon tissue was dissected.The pathological changes of colon were observed by hematoxylin-eosin(HE)and alcian blue-periodic acid-Schiff(AB-PAS)staining.The ultrastructure of colon tissue was observed by transmission electron microscope.Serum levels of interleukin(IL)-6,IL-8,IL-10,IL-1β,tumor necrosis factor α(TNF-α),lipopolysaccharide(LPS)and myeloperoxidase(MPO)were detected by enzyme-linked immunosorbent assay(ELISA).The expression of galectin-3(Gal-3)protein in colon tissue of mice was detected by immunohistochemistry analysis.The expression of MUC2 and the co-localization of NOD-like receptor heat protein domain associated protein 3(NLRP3)and apoptosis-associated speck-like protein(ASC)were detected by immunofluorescence assay.The protein expression of Gal-3,NLRP3,ASC,Caspase-1,IL-1β,IL-18,Occludin,and zonula occludens protein-1(ZO-1)in colon tissue of mice were detected by Western Blot.Results Compared with the control group,the body weight and colon length in the model group were shortened significantly,while the index of colonic weight of mice were increased significantly(P＜0.01).The intestinal mucosal structure of mice was disordered,the microvilli were sparse,the crypt structure was infiltrated by a large number of inflammatory cells,mitochondria were significantly swollen,and goblet cells were obviously decreased.Serum levels of IL-6,IL-8,IL-1β,TNF-α,LPS,and MPO were significantly increased,while IL-10 level was significantly decreased(P＜0.01).The positive expression of MUC2 protein in colon tissue was decreased and the co-localization of NLRP3 and ASC was enhanced.The protein expression of Gal-3,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in colon tissue were significantly increased,and the protein expression of Occludin and ZO-1 were significantly decreased(P＜0.01).Compared with the model group,the body weight and colon length of mice in isoorientin low-and high-dose groups were significantly increased,the index of colonic weight of mice was apparently decreased(P＜0.05,P＜0.01).The structure of intestinal mucosa,microvilli and mitochondria recovered obviously,inflammatory infiltration was alleviated,and goblet cells were increased obviously.Serum levels of IL-6,IL-8,IL-1β,TNF-α,LPS,and MPO were significantly decreased,while IL-10 level was significantly increased(P＜0.05,P＜0.01).The positive expression of MUC2 protein in colon tissue was increased and the co-localization of NLRP3 and ASC was reduced.The protein expression of Gal-3,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in colon tissue were significantly decreased,and the protein expression of Occludin and ZO-1 were significantly increased(P＜0.05,P＜0.01).Conclusion Isoorientin has a great therapeutic effect on DSS-induced UC mice.Its mechanism may be related to the protection of intestinal mucosal barrier by Gal-3/NLRP3/IL-1β signaling pathway.

Objective To explore the activation of tumor necrosis factor-α(TNF-α)signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells.Methods A human proximal renal tubular cell(HK2)model of lentivirus-mediated NPHP1 knockdown(NPHP1KD)was constructed,and the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors CXCL5,CCL20,IL-1β,IL-6 and MCP-1 were detected using RT-qPCR,Western blotting or enzyme-linked immunosorbent assay.A small interfering RNA(siRNA)was transfected in wild-type and NPHP1KDHK2 cells,and the changes in the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors were examined.Results NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α,C/EBPβ,CXCL5,IL-1β,and IL-6(P<0.05),protein expressions of phospho-p38 and C/EBPβ(P<0.05),and IL-6 level in the culture supernatant(P<0.05),and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ(P<0.05).Conclusion TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells,and C/EBPβ may serve as a key transcription factor to mediate these changes.