Objective To observe the effects of Jianpi Huatan Prescription on hepatic cholesterol synthesis in subclinical hypothyroidism(SCH)mice;To discuss its mechanism based on cAMP/CREB/HMGCR signaling pathway.Methods Totally 80 C57BL/6 male mice were randomly divided into control group(10 mice)and modeling group(70 mice),and the modeling group was given methimazole 0.08 mg/(kg·d)in drinking water for 16 weeks to establish a SCH mdoel.The model mice were randomly divided into model group,euthyrox group and Jianpi Huatan Prescription high-,medium-and low-dosage groups,with 10 mice in each group,and were given corresponding drugs for gavage for 6 weeks.HE staining and Oil red O staining were used to observe the morphology and lipid deposition of liver tissue,ELISA was used to detect serum contents of thyroid stimulating hormone(TSH),triiodothyronine(T3),thyroid hormone(T4),total cholesterol(TC),triglycerides(TG),and TC,cyclic adenosine monophosphate(cAMP)in liver tissue,Western blot was used to detect the expressions of protein kinase A(PKA),cyclic adenosine response element binding protein(CREB),p-CREB and 3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR)in liver tissue.Results Compared with the control group,the liver tissue of mice in the model group showed fat vacuoles of different sizes and obvious lipid deposition;the contents of TSH,TC,TG in serum and TC,cAMP in liver tissue significantly increased(P＜0.05,P＜0.01);the protein expressions of PKA,p-CREB and HMGCR significantly increased(P＜0.01).Compared with the model group,lipid deposition in liver tissue and structure of liver cells was improved to varying degrees in euthyrox group and Jianpi Huatan Prescription high-and medium-dosage groups,and the contents of serum TSH,TC,TG,and liver tissue TC,cAMP decreased(P＜0.05,P＜0.01);the expressions of PKA,p-CREB and HMGCR protein in liver tissue increased significantly(P＜0.01).Conclusion Jianpi Huatan Prescription can significantly inhibit hepatic cholesterol synthesis in SCH mice,and improve hepatic fat vacuoles and lipid deposition,and its mechanism may be to reduce TSH levels in SCH mice by regulating the cAMP/CREB/HMGCR signaling pathway.

A long-acting feline ω-interferon fusion protein(FSA-FeIFNω)was designed and its bio-logical function validated.According to the optimization of the sequence of feline serum albumin and feline ω interferon in NCBI,the recombinant plasmid pET-30a(+)-FSA-FeIFNω was con-structed,which was transformed into E.coli BL21(DE3)competent cells,the expression of re-combinant protein FSA-FeIFNω was induced by IPTG,and the expressed inclusion body protein was identified by Western blot,the refolding product was purified by Ni-NTA affinity chromatog-raphy,and the concentration of dialysis and concentrated protein after purification was determined by BCA method.The antiviral activity of recombinant protein was detected by micro-cytopathic in-hibition method in the CRFK/VSV system,the in vitro half-life was detected by 50％mouse plas-ma method,the tumor cell proliferation inhibition activity was detected by MTT method,and anti-tumor activity was detected by mouse melanoma model.The pET-30a(+)-FeIFNω and pET-30a(+)-FSA-FeIFNω expression vectors were successfully constructed,and 87 kDa recombinant FSA-FeIFNω protein was obtained in E.coli,with a purified protein purity of 95％,with a concen-tration of 1 g/L,and the biological activity was 2.56 × 106 IU/mg,the plasma half-life was significantly prolonged(＞24 h),and the half-inhibitory concentration IC50 of B16-F10 in mouse melanoma cells was 56.01 mg/L.The FSA-FeIFNω group significantly inhibited tumor growth,and the treatment effect was better than that of the control group and other experimental groups.The recombinant FSA-FeIFNω protein obtained in this study had long-acting effect and good biological activity.

This study aims to construct a recombinant vaccine containing the tetanus toxin C frag-ment(HC)with a built-in adjuvant of metallothionein 3(MT-3)and evaluate its immune effect in mice.The gene sequences of MT-3 and HC were fused via a linker to create the pET30a-MT-3-HC recombinant plasmid,which was then transformed into E.coli BL21(DE3)competent cells.The re-combinant plasmid was confirmed through double enzyme digestion.The M3C recombinant protein expression was induced,identified by SDS-PAGE and Western blot,and purified using Ni-NTA af-finity chromatography.Five groups of vaccines,including PBS,HC,TT(tetanus toxoid),M3C,and M3C+CpG(composite adjuvant),were administered to mice via intramuscular injection at 7-day intervals for three immunizations.Blood samples were periodically collected from the tail vein.ELISA was used to measure changes in specific IgG antibody titers in the serum,and on day 28,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3,and IgM)and cytokine levels(IL-4,IFN-γ)were also measured.The results demonstrated that the pET30a-MT-3-HC recombinant plasmid was cor-rectly constructed,and the M3C recombinant protein was highly expressed in the supernatant fol-lowing ultrasonic disruption of the induced bacterial culture,with a single band observed post-puri-fication.ELISA results showed that the titer of specific IgG antibodies in the M3C+CpG group peaked at 3.54×105 14 days after the third immunization,which was 141 times,70 times,and 6.6 times higher than that in HC,TT,and TT groups,respectively.Antibody subtype analysis revealed significantly higher specific antibody subtype titers in the M3C and M3C+CpG groups compared to the PBS,HC,and TT groups(P＜0.05),with the M3 C group showing an IgG1/IgG2a ratio greater than 1,and the M3C+CpG group having an IgG1/IgG2a ratio of approximately 1.Serum concentrations of IFN-γ and IL-4 in the M3C and M3C+CpG groups were also significantly higher than those in the other experimental groups(P＜0.05).These results showed that the recombinant antigen containing MT-3 built-in adjuvant and tetanus toxin C fragment was successfully expressed and showed strong immunogenicity,which laid the experimental foundation for the development of this recombinant vaccine.

Objective:To observe the therapeutic effects of catheter balloon dilation and high-intensity Shaker training on cri-copharyngeal achalasia after brainstem stroke and the effect on post-stroke aspiration pneumonia.Method:Fifty patients with swallowing disorders after brainstem stroke admitted to Yangfangdian Hospital and Wangjing Hospital between July 2022 and December 2022 were selected for this non-blinded clinical trial.The pa-tients were divided into the control group(n=25)and the experimental group(n=25)according to the order of ad-mission and receipt of balloon dilation.The control group was treated with conventional swallowing therapy and increased the intensity of Shaker training,while the experimental group was treated with additional transoral bal-loon dilatation.The changes of swallowing function and incidence of aspiration pneumonia were compared using videofluoroscopic swallowing study(VFSS),the standard swallowing function assessment scale(SSA),Rosenbek infiltration/misaspiration scale,feeding function and the incidence of aspiration pneumonia.The self-assessment de-pression scale(SDS)and anxiety self-assessment anxiety scale(SAS)were used to assess the changes of pa-tients'psychological status.Result:There was no statistically significant differences in swallowing function and psychological status between the two groups before treatment(P>0.05).After 8 weeks of treatment,both groups showed improvement in SSA,VFSS and Rosenbek scores(P<0.05),and significant reductions in SAS and SDS,with significant improvement in feeding function.The improvement in swallowing and psychological status of both groups was better than pre-treatment,with significantly higher in the experimental group than the control group(P<0.05).Conclusion:Both the rehabilitation training method of swallowing with increasing the intensity of Shaker training and balloon dilation are effective in the recovery of swallowing disorder,but the combined treatment is more ef-fective.Both group showed improvements in Rosenbek score and reduced incidence of aspiration after treatment,indicating that the rehabilitation training with increasing intensity of Shaker training and balloon dilation can effec-tively reduce the incidence of aspiration pneumonia,and the combined treatment was more effective.

To develop a novel immunocastration vaccine for animals,researchers designed and syn-thesized the recombinant plasmid pET-30a-SF which could express the recombinant protein SF.This protein was then conjugated in vitro with the synthetic peptide STGP to prepare the SF-STGP nanoparticle vaccine,and its immunocastration effect on mice was studied.The Spy Catcher and ferritin amino acid sequences were connected via GGGGS,and after codon optimization for E.coli,the recombinant plasmid pET-30a-SF was constructed and transformed into E.coli for in-duced expression.The recombinant protein SF was purified using Ni-column affinity chromatogra-phy and characterized.The peptide STGP,composed of Spy Tag,GnRH,and PADRE connected by GGGS,was conjugated with the recombinant protein SF in vitro.The self-assembled nanoparticles were observed using transmission electron microscopy(TEM)and dynamic light scattering(DLS).The prepared SF-STGP nanoparticles were mixed with MONTANIDE ISA 206 VG at a 1∶1 ratio to form the vaccine,which was then subcutaneously injected into male BALB/c mice for immunocastration evaluation.The recombinant protein SF showed the highest soluble expression when induced at 18 ℃ with 0.25 mmol/L IPTG for 14 h,and the maximum conjugation efficiency with STGP was achieved at a 1∶8 molar ratio.TEM and DLS analyses revealed that both the re-combinant protein SF and SF-STGP could self-assemble into nanoparticles with average diameters of 16.2 nm and 17.8 nm,respectively.Mouse immunization results demonstrated that the SF-STGP nanoparticle vaccine generated specific GnRH antibodies after the first immunization,with the spe-cific antibody D45o reaching its peak at the 10 th week.The SF-STGP+ISA 206 immunization group showed a peak D450 value of 2.8,and the specific antibody levels in all immunization groups were significantly higher than those in the control group(P＜0.05).Additionally,the SF-STGP nanoparticle vaccine effectively reduced serum testosterone levels in mice,with the testosterone concentration in the immunization groups being significantly lower than that in the control group(P＜0.05).Compared to the control group,the immunization group exhibits testicular atrophy.The constructed SF-STGP nanoparticle vaccine proves to be a highly effective immunogen,capable of inducing testicular atrophy and reducing gonadal hormone concentrations,demonstrating excellent castration effects.This study provides new insights into immunocastration vaccines for mammals.

Objective To observe the effects of Jianpi Huatan Prescription on hepatic cholesterol synthesis in subclinical hypothyroidism(SCH)mice;To discuss its mechanism based on cAMP/CREB/HMGCR signaling pathway.Methods Totally 80 C57BL/6 male mice were randomly divided into control group(10 mice)and modeling group(70 mice),and the modeling group was given methimazole 0.08 mg/(kg·d)in drinking water for 16 weeks to establish a SCH mdoel.The model mice were randomly divided into model group,euthyrox group and Jianpi Huatan Prescription high-,medium-and low-dosage groups,with 10 mice in each group,and were given corresponding drugs for gavage for 6 weeks.HE staining and Oil red O staining were used to observe the morphology and lipid deposition of liver tissue,ELISA was used to detect serum contents of thyroid stimulating hormone(TSH),triiodothyronine(T3),thyroid hormone(T4),total cholesterol(TC),triglycerides(TG),and TC,cyclic adenosine monophosphate(cAMP)in liver tissue,Western blot was used to detect the expressions of protein kinase A(PKA),cyclic adenosine response element binding protein(CREB),p-CREB and 3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR)in liver tissue.Results Compared with the control group,the liver tissue of mice in the model group showed fat vacuoles of different sizes and obvious lipid deposition;the contents of TSH,TC,TG in serum and TC,cAMP in liver tissue significantly increased(P＜0.05,P＜0.01);the protein expressions of PKA,p-CREB and HMGCR significantly increased(P＜0.01).Compared with the model group,lipid deposition in liver tissue and structure of liver cells was improved to varying degrees in euthyrox group and Jianpi Huatan Prescription high-and medium-dosage groups,and the contents of serum TSH,TC,TG,and liver tissue TC,cAMP decreased(P＜0.05,P＜0.01);the expressions of PKA,p-CREB and HMGCR protein in liver tissue increased significantly(P＜0.01).Conclusion Jianpi Huatan Prescription can significantly inhibit hepatic cholesterol synthesis in SCH mice,and improve hepatic fat vacuoles and lipid deposition,and its mechanism may be to reduce TSH levels in SCH mice by regulating the cAMP/CREB/HMGCR signaling pathway.

Objective:To enhance the understanding of clinical doctors on the concurrence of Sj?gren′s syndrome (SS) and myasthenia gravis (MG), provide reference forclinical diagnosis and treatment.Methods:The medical record of SS、MG patient Taian Central hospital in october 2021 was reviewed and a retrospective analysis was conducted. Based on the key words, the main databases domestic and abroad from Jan 1973 to April 2024. The cases reported in the literature were searched out and merged with our case and analyzed by SPSS 27.0. The measurement data of normal distribution were expressed by mean standard deviation ± s, and the measurement data of abnormal distribution were expressed by M( Q1, Q3). Counting data were expressed as examples and percentages (%) . Results:Twenty-one patients with SS complicated with MG were reviewed. Among them, 19(90.5%) patients were female. The average age was (47.6 ± 3.1) years, and the median time from onset to diagnosis was 24 (11, 78) months. There were 11 patients (52.4%) with myasthenia gravis as the initial presentation. The main clinical manifestations of patients with myasthenia muscle fatigue (80.9%, 17/21), gravis include fatigue ptosis (71.4%, 15/21), diplopia, blurred vision (57.1%, 12/21), dysphagia (33.3%, 7/21), joint pain (33.3%, 7/21), odysarthria, and unclear speech (23.8%, 5/21), hair loss (14.3%, 3/21), and swelling of the excocrine glands(9.5%, 2/21). Eighteen patients (85.7%, 18/21) showed positive anti acetylcholine receptor antibodies. Sixteen patients (76.2%) were positive for ANA, and 14 patients (66.8%) were positive for anti SSA or (and) anti SSB antibodies. Four patients (19.0%) also had other autoimmune diseases (Hashimoto′s thyroiditis, rheumatoid arthritis, optic neuritis). In terms of treatment, cholinesterase inhibitors were the most common treatment measure, applied to 18 patients (85.7%), followed by glucocorticoid therapy, and applied to 14 patients (66.8%). In addition, 7 patients (33.3%) received immunosuppressive agents therapy, 5 patients (23.8%) underwent thymectomy, 2 patients (9.5%) received intravenous human immunoglobulin injection, and 1 patient (4.8%) underwent plasma exchange. All patients showed improvement after treatment.Conclusion:SS and MG are both autoimmune diseases, and their coexistence is rare. Clinicans should be aware of this rare association. Early diagnosis is crucial for the treatment and prognosis of patients,and requiresa comprehensive assessment of clinical symptoms, laboratory tests, and auto-antibody test results.

To develop a novel immunocastration vaccine for animals,researchers designed and syn-thesized the recombinant plasmid pET-30a-SF which could express the recombinant protein SF.This protein was then conjugated in vitro with the synthetic peptide STGP to prepare the SF-STGP nanoparticle vaccine,and its immunocastration effect on mice was studied.The Spy Catcher and ferritin amino acid sequences were connected via GGGGS,and after codon optimization for E.coli,the recombinant plasmid pET-30a-SF was constructed and transformed into E.coli for in-duced expression.The recombinant protein SF was purified using Ni-column affinity chromatogra-phy and characterized.The peptide STGP,composed of Spy Tag,GnRH,and PADRE connected by GGGS,was conjugated with the recombinant protein SF in vitro.The self-assembled nanoparticles were observed using transmission electron microscopy(TEM)and dynamic light scattering(DLS).The prepared SF-STGP nanoparticles were mixed with MONTANIDE ISA 206 VG at a 1∶1 ratio to form the vaccine,which was then subcutaneously injected into male BALB/c mice for immunocastration evaluation.The recombinant protein SF showed the highest soluble expression when induced at 18 ℃ with 0.25 mmol/L IPTG for 14 h,and the maximum conjugation efficiency with STGP was achieved at a 1∶8 molar ratio.TEM and DLS analyses revealed that both the re-combinant protein SF and SF-STGP could self-assemble into nanoparticles with average diameters of 16.2 nm and 17.8 nm,respectively.Mouse immunization results demonstrated that the SF-STGP nanoparticle vaccine generated specific GnRH antibodies after the first immunization,with the spe-cific antibody D45o reaching its peak at the 10 th week.The SF-STGP+ISA 206 immunization group showed a peak D450 value of 2.8,and the specific antibody levels in all immunization groups were significantly higher than those in the control group(P＜0.05).Additionally,the SF-STGP nanoparticle vaccine effectively reduced serum testosterone levels in mice,with the testosterone concentration in the immunization groups being significantly lower than that in the control group(P＜0.05).Compared to the control group,the immunization group exhibits testicular atrophy.The constructed SF-STGP nanoparticle vaccine proves to be a highly effective immunogen,capable of inducing testicular atrophy and reducing gonadal hormone concentrations,demonstrating excellent castration effects.This study provides new insights into immunocastration vaccines for mammals.

Objective:To observe the therapeutic effects of catheter balloon dilation and high-intensity Shaker training on cri-copharyngeal achalasia after brainstem stroke and the effect on post-stroke aspiration pneumonia.Method:Fifty patients with swallowing disorders after brainstem stroke admitted to Yangfangdian Hospital and Wangjing Hospital between July 2022 and December 2022 were selected for this non-blinded clinical trial.The pa-tients were divided into the control group(n=25)and the experimental group(n=25)according to the order of ad-mission and receipt of balloon dilation.The control group was treated with conventional swallowing therapy and increased the intensity of Shaker training,while the experimental group was treated with additional transoral bal-loon dilatation.The changes of swallowing function and incidence of aspiration pneumonia were compared using videofluoroscopic swallowing study(VFSS),the standard swallowing function assessment scale(SSA),Rosenbek infiltration/misaspiration scale,feeding function and the incidence of aspiration pneumonia.The self-assessment de-pression scale(SDS)and anxiety self-assessment anxiety scale(SAS)were used to assess the changes of pa-tients'psychological status.Result:There was no statistically significant differences in swallowing function and psychological status between the two groups before treatment(P>0.05).After 8 weeks of treatment,both groups showed improvement in SSA,VFSS and Rosenbek scores(P<0.05),and significant reductions in SAS and SDS,with significant improvement in feeding function.The improvement in swallowing and psychological status of both groups was better than pre-treatment,with significantly higher in the experimental group than the control group(P<0.05).Conclusion:Both the rehabilitation training method of swallowing with increasing the intensity of Shaker training and balloon dilation are effective in the recovery of swallowing disorder,but the combined treatment is more ef-fective.Both group showed improvements in Rosenbek score and reduced incidence of aspiration after treatment,indicating that the rehabilitation training with increasing intensity of Shaker training and balloon dilation can effec-tively reduce the incidence of aspiration pneumonia,and the combined treatment was more effective.

A long-acting feline ω-interferon fusion protein(FSA-FeIFNω)was designed and its bio-logical function validated.According to the optimization of the sequence of feline serum albumin and feline ω interferon in NCBI,the recombinant plasmid pET-30a(+)-FSA-FeIFNω was con-structed,which was transformed into E.coli BL21(DE3)competent cells,the expression of re-combinant protein FSA-FeIFNω was induced by IPTG,and the expressed inclusion body protein was identified by Western blot,the refolding product was purified by Ni-NTA affinity chromatog-raphy,and the concentration of dialysis and concentrated protein after purification was determined by BCA method.The antiviral activity of recombinant protein was detected by micro-cytopathic in-hibition method in the CRFK/VSV system,the in vitro half-life was detected by 50％mouse plas-ma method,the tumor cell proliferation inhibition activity was detected by MTT method,and anti-tumor activity was detected by mouse melanoma model.The pET-30a(+)-FeIFNω and pET-30a(+)-FSA-FeIFNω expression vectors were successfully constructed,and 87 kDa recombinant FSA-FeIFNω protein was obtained in E.coli,with a purified protein purity of 95％,with a concen-tration of 1 g/L,and the biological activity was 2.56 × 106 IU/mg,the plasma half-life was significantly prolonged(＞24 h),and the half-inhibitory concentration IC50 of B16-F10 in mouse melanoma cells was 56.01 mg/L.The FSA-FeIFNω group significantly inhibited tumor growth,and the treatment effect was better than that of the control group and other experimental groups.The recombinant FSA-FeIFNω protein obtained in this study had long-acting effect and good biological activity.