Neurological diseases have high morbidity and disability rates， posing a severe threat to human health. Cli nical manifestations include motor， sensory， cognitive and conscious disorders. Chaihu jia longgu muli decoction is derived from Treatise on Febrile Diseases ， with the effects of harmonizing Shaoyang， activating Yang and clearing heat， and tranquilizing the mind. This paper systematically reviews the research progress in clinical application and mechanism of Chaihu jia longgu muli decoction in the field of neurological diseases. It has been found that the decoction shows favorable efficacy in various neurological diseases such as insomnia， depression， epilepsy， vertigo， migraine and vascular dementia. The specific mechanisms are related to regulating neurotransmitter levels， repairing neuronal function， alleviating neuroinflammation， improving mitochondrial dysfunction and regulating intestinal flora. In the future， standardized prospective follow-up cohorts should be established， and core outcome indicators should be clearly defined to strengthen the evidence base. Furthermore， multidisciplinary research should be leveraged to expand the therapeutic value of Chaihu jia longgu muli decoction in the management of neurological diseases.

Objective:To establish an accurate, stable, and convenient Taka amylase enzymolysis ion-selective electrode method for determination of trace fluoride in Mongolian milk tea powder.Methods:The samples of instant Mongolian milk tea powder were heated and dried at 50 ℃, 1.0 g of the sample was accurately weighed and dissolved in pure water. The sample solution, Taka amylase, and total ion strength buffer solution were mixed and enzymatically hydrolyzed in a 55 ℃ water bath for 60 minutes. After cooling, filtration was carried out. The clarified liquid was collected and the fluoride content was detected by the ion-selective electrode method, and the method validation test was conducted.Results:When the standard linear concentration range was 0.2 to 5.0 mg/L, the correlation coefficient ranged from 0.999 5 to 0.999 9. The detection limit of the method was 0.006 mg/kg. The relative standard deviations ( RSD) for detecting high, medium, and low concentration samples were 0.8%, 1.9%, and 2.1%, respectively. The recovery rates ranged from 98% to 101%, 95% to 101%, and 101% to 107%, respectively. This method simultaneously measured the same batch of samples with the second method of the "National Food Safety Standard - Determination of Fluorine in Foods" (GB 5009.18-2025) - the ion-selective electrode method. The results were (23.1 ± 7.7) and (22.5 ± 7.6) mg/kg, respectively. There was no statistically significant difference between the two methods ( t = 2.01, P = 0.066). Conclusion:The Taka amylase enzymolysis ion-selective electrode method has good reproducibility and high accuracy, making it suitable for determination of trace fluoride in Mongolian milk tea powder.

Objective:To assess the efficacy and safety of Tacrolimus（TAC）in combination with glucocorticosteroid（GC）for treating IgA vasculitis nephritis（IgAVN）in children.Methods:A retrospective analysis was conducted on pediatric patients who were diagnosed with IgAVN from January 2015 to January 2022 in Children's Hospital of Hebei Province.The patients presented with nephrotic-range proteinuria or persistent urine protein（>0.5g/24 h）despite adequate glucocorticoid and other treatments in patients who do not reach massive proteinuria levels.They were treated with TAC combined with GC. The following laboratory parameters were obtained for outcome assessment: 24-hour urinary protein excretion, serum albumin, serum creatinine levels, and fasting blood glucose measurements. The efficacy and adverse reactions of TAC were summarized.Results:A total of 97 children (55 males and 42 females) were included. The average age of diagnosis of IgA vasculitis was (8.65±2.46) years, and 95.9% of the children developed renal involvement within 30 days after diagnosis. Pathological examination of renal puncture: 5 cases of grade Ⅱa, 2 cases of grade Ⅱb, 31 cases of grade Ⅲa, 57 cases of grade Ⅲb, and 2 cases of grade Ⅳb.Remission rate at 3 months was 96.9%（94/97）.Three patients failed to achieve clinical remission who were treaed with other immunosuppressants.After 1, 3, 6 and 12 months of TAC treatment, the urine protein levels of 94 children were lower than those before treatment, and the differences were statistically significant ( P < 0.05), showing a gradual downward trend. Serum albumin levels were higher than those before treatment, and the differences were statistically significant ( P < 0.05), showing a gradual upward trend.After 3 months and 6 months of TAC treatment, the serum creatinine and fasting blood glucose of the children increased. With the remission of the disease, TAC dosage decreased, the mean values of serum creatinine and fasting blood glucose decreased after 12 months of treatment.The average treatment time of TAC was (10.8±2.6) months, the average follow-up time was (3.33±1.56) years, and the longest follow-up time was 8 years. During the follow-up period, there were no serious adverse reactions such as gastrointestinal discomfort, liver function damage and severe infection. After stopping GC and TAC treatment, 80 children got sustained remission. Conclusion:The combination of TAC and GC has been proved to be effective in treating IgAVN in children.The overall effective rate is high,and clinical remission can be achieved quickly with relatively mild adverse reactions.

Objective:To analyze a Chinese pedigree with Hereditary coagulation factor Ⅻ (FⅫ) deficiency duo to variants of F12 gene and explore its molecular pathogenesis. Methods:A patient who underwent laparoscopic cystectomy at the Department of Gynecology of the First Affiliated Hospital of Wenzhou Medical University in June 2012 was selected as the study subject. Coagulation factor indexes of the proband and her family members (5 individuals from three generations) were determined. All exons, flanking sequences, 5′ and 3′ untranslated regions of the F12 gene of the proband and her family members were analyzed by direct sequencing. Three bioinformatics software was used to analyze the conservation, pathogenicity and protein model of the variant. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No. 2012-17). Results:The activated partial thromboplastin time (APTT), FⅫ activity (FⅫ: C) and FⅫ antigen (FⅫ: Ag) of the proband was 180.0 s, 1.0% and 2.1%, respectively. DNA sequencing revealed that she has harbored compound heterozygous variants of the F12 gene, namely c. 712_713insT (p.Cys238Leufs *73) in exon 8 and c. 1561G>A (p.Glu521Lys) in exon 13. Her mother and younger son were heterozygous for the p. Cys238Leufs*73 variant, while her older son was heterozygous for the p. Glu521Lys variant. Bioinformatic analysis suggested that Cys238 is highly conserved and p. Cys238Leufs*73 is a pathogenic variant, which eventually resulted in a truncated protein. Conclusion:The c. 712_713insT and c. 1561G>A compound heterozygous variants of the F12 gene probably underlay the decreased FⅫ level in this pedigree, among which c. 712_713insT (NM_000505) was unreported previously.

Objective:To investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C (PROC) genes through laboratory phenotype analysis, genetic mutation analysis, and in vitro expression experiments.Methods:Two probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University. Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C (PC) activity (PC∶A), PC antigen (PC∶Ag) levels, and other relevant coagulation parameters. The anticoagulant capacity was assessed using the thrombin generation test (TGT). The mutation sites of the PROC gene were identified using direct DNA sequencing. Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene. PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids. Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected. Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene. Enzyme-linked immunosorbent assay, Western blot, and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein.Results:Probands 1 and 2 exhibited PC∶A levels of 35% and 40% and PC∶Ag levels of 44% and 39%, with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L, respectively. Meanwhile, other coagulation parameters revealed no significant abnormalities. TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers. Sequencing analysis revealed heterozygous missense mutations c. 833T>C (p. Leu278Pro) in proband 1 and c. 1330T>C (p. Trp444Arg) in proband 2 within exon 9 of the PROC gene. Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species. Pathogenicity analysis indicated that both p. Leu278Pro and p. Trp444Arg mutations are deleterious. Protein modeling analysis demonstrated that both mutations induce structural alterations in the protein. In vitro expression experiments revealed that compared with the wild-type, both p. Leu278Pro and p. Trp444Arg mutations showed no significant differences in the mRNA expression level of the PC protein. However, both mutations caused significantly lower PC∶Ag content and protein expression levels in the cell culture supernatant compared with the wild-type, whereas higher levels were observed in the cell culture lysate. This indicates the association of both mutations with the secretion function of the PC protein.Conclusion:The heterozygous missense mutations p. Leu278Pro and p. Trp444Arg in exon 9 of the PROC gene in both probands are associated with decreased PC levels.

OBJECTIVE: To analyze a Chinese pedigree with Hereditary coagulation factor Ⅻ (FⅫ) deficiency duo to variants of F12 gene and explore its molecular pathogenesis. METHODS: A patient who underwent laparoscopic cystectomy at the Department of Gynecology of the First Affiliated Hospital of Wenzhou Medical University in June 2012 was selected as the study subject. Coagulation factor indexes of the proband and her family members (5 individuals from three generations) were determined. All exons, flanking sequences, 5' and 3' untranslated regions of the F12 gene of the proband and her family members were analyzed by direct sequencing. Three bioinformatics software was used to analyze the conservation, pathogenicity and protein model of the variant. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No. 2012-17). RESULTS: The activated partial thromboplastin time (APTT), FⅫ activity (FⅫ:C) and FⅫ antigen (FⅫ:Ag) of the proband was 180.0 s, 1.0% and 2.1%, respectively. DNA sequencing revealed that she has harbored compound heterozygous variants of the F12 gene, namely c.712_713insT (p.Cys238Leufs *73) in exon 8 and c.1561G>A (p.Glu521Lys) in exon 13. Her mother and younger son were heterozygous for the p.Cys238Leufs*73 variant, while her older son was heterozygous for the p.Glu521Lys variant. Bioinformatic analysis suggested that Cys238 is highly conserved and p.Cys238Leufs*73 is a pathogenic variant, which eventually resulted in a truncated protein. CONCLUSION The c.712_713insT and c.1561G>A compound heterozygous variants of the F12 gene probably underlay the decreased FⅫ level in this pedigree, among which c.712_713insT (NM_000505) was unreported previously.
Adult ; Female ; Humans ; Male ; Middle Aged ; Base Sequence ; China ; Factor XII/genetics* ; Heterozygote ; Mutation ; Pedigree ; Factor XII Deficiency/genetics* ; East Asian People

OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

Objective:To establish an accurate, stable, and convenient Taka amylase enzymolysis ion-selective electrode method for determination of trace fluoride in Mongolian milk tea powder.Methods:The samples of instant Mongolian milk tea powder were heated and dried at 50 ℃, 1.0 g of the sample was accurately weighed and dissolved in pure water. The sample solution, Taka amylase, and total ion strength buffer solution were mixed and enzymatically hydrolyzed in a 55 ℃ water bath for 60 minutes. After cooling, filtration was carried out. The clarified liquid was collected and the fluoride content was detected by the ion-selective electrode method, and the method validation test was conducted.Results:When the standard linear concentration range was 0.2 to 5.0 mg/L, the correlation coefficient ranged from 0.999 5 to 0.999 9. The detection limit of the method was 0.006 mg/kg. The relative standard deviations ( RSD) for detecting high, medium, and low concentration samples were 0.8%, 1.9%, and 2.1%, respectively. The recovery rates ranged from 98% to 101%, 95% to 101%, and 101% to 107%, respectively. This method simultaneously measured the same batch of samples with the second method of the "National Food Safety Standard - Determination of Fluorine in Foods" (GB 5009.18-2025) - the ion-selective electrode method. The results were (23.1 ± 7.7) and (22.5 ± 7.6) mg/kg, respectively. There was no statistically significant difference between the two methods ( t = 2.01, P = 0.066). Conclusion:The Taka amylase enzymolysis ion-selective electrode method has good reproducibility and high accuracy, making it suitable for determination of trace fluoride in Mongolian milk tea powder.

Objective:To assess the efficacy and safety of Tacrolimus（TAC）in combination with glucocorticosteroid（GC）for treating IgA vasculitis nephritis（IgAVN）in children.Methods:A retrospective analysis was conducted on pediatric patients who were diagnosed with IgAVN from January 2015 to January 2022 in Children's Hospital of Hebei Province.The patients presented with nephrotic-range proteinuria or persistent urine protein（>0.5g/24 h）despite adequate glucocorticoid and other treatments in patients who do not reach massive proteinuria levels.They were treated with TAC combined with GC. The following laboratory parameters were obtained for outcome assessment: 24-hour urinary protein excretion, serum albumin, serum creatinine levels, and fasting blood glucose measurements. The efficacy and adverse reactions of TAC were summarized.Results:A total of 97 children (55 males and 42 females) were included. The average age of diagnosis of IgA vasculitis was (8.65±2.46) years, and 95.9% of the children developed renal involvement within 30 days after diagnosis. Pathological examination of renal puncture: 5 cases of grade Ⅱa, 2 cases of grade Ⅱb, 31 cases of grade Ⅲa, 57 cases of grade Ⅲb, and 2 cases of grade Ⅳb.Remission rate at 3 months was 96.9%（94/97）.Three patients failed to achieve clinical remission who were treaed with other immunosuppressants.After 1, 3, 6 and 12 months of TAC treatment, the urine protein levels of 94 children were lower than those before treatment, and the differences were statistically significant ( P < 0.05), showing a gradual downward trend. Serum albumin levels were higher than those before treatment, and the differences were statistically significant ( P < 0.05), showing a gradual upward trend.After 3 months and 6 months of TAC treatment, the serum creatinine and fasting blood glucose of the children increased. With the remission of the disease, TAC dosage decreased, the mean values of serum creatinine and fasting blood glucose decreased after 12 months of treatment.The average treatment time of TAC was (10.8±2.6) months, the average follow-up time was (3.33±1.56) years, and the longest follow-up time was 8 years. During the follow-up period, there were no serious adverse reactions such as gastrointestinal discomfort, liver function damage and severe infection. After stopping GC and TAC treatment, 80 children got sustained remission. Conclusion:The combination of TAC and GC has been proved to be effective in treating IgAVN in children.The overall effective rate is high,and clinical remission can be achieved quickly with relatively mild adverse reactions.

Objective:To analyze a Chinese pedigree with Hereditary coagulation factor Ⅻ (FⅫ) deficiency duo to variants of F12 gene and explore its molecular pathogenesis. Methods:A patient who underwent laparoscopic cystectomy at the Department of Gynecology of the First Affiliated Hospital of Wenzhou Medical University in June 2012 was selected as the study subject. Coagulation factor indexes of the proband and her family members (5 individuals from three generations) were determined. All exons, flanking sequences, 5′ and 3′ untranslated regions of the F12 gene of the proband and her family members were analyzed by direct sequencing. Three bioinformatics software was used to analyze the conservation, pathogenicity and protein model of the variant. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No. 2012-17). Results:The activated partial thromboplastin time (APTT), FⅫ activity (FⅫ: C) and FⅫ antigen (FⅫ: Ag) of the proband was 180.0 s, 1.0% and 2.1%, respectively. DNA sequencing revealed that she has harbored compound heterozygous variants of the F12 gene, namely c. 712_713insT (p.Cys238Leufs *73) in exon 8 and c. 1561G>A (p.Glu521Lys) in exon 13. Her mother and younger son were heterozygous for the p. Cys238Leufs*73 variant, while her older son was heterozygous for the p. Glu521Lys variant. Bioinformatic analysis suggested that Cys238 is highly conserved and p. Cys238Leufs*73 is a pathogenic variant, which eventually resulted in a truncated protein. Conclusion:The c. 712_713insT and c. 1561G>A compound heterozygous variants of the F12 gene probably underlay the decreased FⅫ level in this pedigree, among which c. 712_713insT (NM_000505) was unreported previously.