Acupoint application therapy alleviates pain by regulating immune function through inhibiting TLR4/MyD88/NF-κB p65 signaling in a primary dysmenorrhea rat model. / 穴位贴敷通过TLR4/MyD88/NF-κBé€šè·¯è°ƒèŠ‚åŽŸå‘æ€§ç—›ç»å¤§é¼ çš„å ç–«åŠŸèƒ½.

OBJECTIVES: To investigate the effects of graphene-based warm uterus acupoint paste on uterine Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway and Th1/Th2 immune balance in primary dysmenorrhea ( PD ) model rats, so as to reveal its immunological mechanisms of relieving dysmenorrhea. METHODS: Thirty SD female rats were randomly divided into 3 groups：normal group, model group and acupoint paste group, with 10 rats in each group. PD rat model was established by subcutaneous injection of estradiol benzoate for 10 consecutive days. At the same time of modeling, graphene-based warm uterus acupoint paste was applied to the acupoints of "Guanyuan" (CV4), bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6) of rats in the acupoint paste group. The application was continuously applied once daily for 10 d, 5 h each time. On the 11th day, oxytocin was injected intraperitoneally to observe the writhing latency, writhing times within 30 min and writhing score of rats in each group. The spleen and thymus indexes were calculated. The pathological changes of spleen and thymus tissue were observed after HE staining. The contents of serum immunoglobulin (Ig) A, IgG, tumor necrosis factor-α (TNF-α), interleukin (IL)-2, interferon-γ (IFN-γ), IL-4 and IL-10 were detected by ELISA . The protein and mRNA expression levels of TLR4, MyD88 and NF-κB p65 in rat uterine tissue were detected by Western blot and real-time quantitative PCR, respectively. RESULTS: Compared with the normal group, the writhing times and writhing scores within 30 min of rats in the model group were significantly increased(P<0.001), and the rats showed writhing reaction (P<0.01). The spleen index and thymus index were significantly decreased(P<0.01, P<0.05). The spleen and thymus had obvious pathological changes. The contents of IgA, IgG, TNF-α, IL-2 and IFN-γ in serum were significantly increased, while the contents of serum IL-4 and IL-10 were significantly decreased(P<0.001, P<0.01). The expression levels of TLR4, MyD88, NF-κB p65 protein and corresponding mRNA in uterine tissue were significantly increased(P<0.001). Following intervention, compared with the model group, the writhing latency time of rats in the acupoint paste group was prolonged, and the writhing times and writhing scores within 30 min were significantly decreased (P<0.001). The spleen index and thymus index were significantly increased(P<0.01, P<0.05). The pathological changes of spleen and thymus were improved. The contents of serum IgA, IgG, TNF-α, IL-2 and IFN-γ were significantly decreased, while the contents of IL-4 and IL-10 were significantly increased(P<0.001, P<0.05, P<0.01). The expression of TLR4, MyD88, NF-κB p65 protein and the corresponding mRNA levels in uterine tissue were decreased(P<0.001, P<0.01). CONCLUSIONS: Graphene-based warm uterus acupoint paste can regulate the immune balance of Th1/ Th2 by regulating TLR4/ MyD88/ NF-κB p65 signaling pathway, repair the pathological damage of immune tissue, improve immune function, and effectively relieve the pain symptoms of PD rats.

A mouse model for X-linked Alport syndrome induced by Del-ATGG in the Col4a5 gene.

Alport syndrome (AS) is an inherited glomerular basement membrane (GBM) disease leading to end-stage renal disease (ESRD). X-linked AS (XLAS) is caused by pathogenic variants in the COL4A5 gene. Many pathogenic variants causing AS have been detected, but the genetic modifications and pathological alterations leading to ESRD have not been fully characterized. In this study, a novel frameshift variant c.980_983del ATGG in the exon 17 of the COL4A5 gene detected in a patient with XLAS was introduced into a mouse model in by CRISPR/Cas9 system. Through biochemical urinalysis, histopathology, immunofluorescence, and transmission electron microscopy (TEM) detection, the clinical manifestations and pathological alterations of Del-ATGG mice were characterized. From 16 weeks of age, obvious proteinuria was observed and TEM showed typical alterations of XLAS. The pathological changes included glomerular atrophy, increased monocytes in renal interstitial, and the absence of type IV collagen α5. The expression of Col4a5 was significantly decreased in Del-ATGG mouse model. Transcriptomic analysis showed that differentially expressed genes (DEGs) accounted for 17.45% (4,188/24003) of all genes. GO terms indicated that the functions of identified DEGs were associated with cell adhesion, migration, and proliferation, while KEGG terms found enhanced the degradation of ECM, amino acid metabolism, helper T-cell differentiation, various receptor interactions, and several important pathways such as chemokine signaling pathway, NF-kappa B signaling pathway, JAK-STAT signaling pathway. In conclusion, a mouse model with a frameshift variant in the Col4a5 gene has been generated to demonstrate the biochemical, histological, and pathogenic alterations related to AS. Further gene expression profiling and transcriptomic analysis revealed DEGs and enriched pathways potentially related to the disease progression of AS. This Del-ATGG mouse model could be used to further define the genetic modifiers and potential therapeutic targets for XLAS treatment.

Comparative Functional Analysis in vitro of 2 COL4A5 Splicing Mutations at the Same Site in 2 Unrelated Alport Syndrome Chinese Families.

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.

[46,XY DSD induced by a novel mutation c.458T>C (p.Leu153Pro) of the LHCGR gene: A case report and review of the literature].

OBJECTIVE: To investigate the clinical characteristics and pathogenic basis of a case of 46, XY disorders of sex development (DSD) and analyze the relationship of the missense mutation with the phenotype of the LHCGR gene. METHODS: We analyzed the causative gene mutation by next-generation high-throughput sequencing (HTS) and confirmed it by Sanger sequencing. We detected the effect of the mutation on the splicing function by minigene assay, evaluated its pathogenicity using the ANNOVAR mutation annotation software, and analyzed the relationship of the missense mutation and the phenotype of the LHCGR gene via literature review and data mining. RESULTS: A homozygous mutation of C.458T>C (p.Leu153Pro) was detected in the last base of exon5 of the LHCGR gene in the 46,XY DSD patient, which was a new mutation not reported previously. The mother of the patient was a heterozygous carrier of the mutation. Minigene assay indicated that c.458T>C (p.Leu153Pro) did not affect the splicing function. The mutation was shown to be pathogenic by ANNOVAR software analysis and presumed inactive, possibly affecting its binding with the ligand and leading to type-I Leydig cell hypoplasia (LCH). Literature review and data mining showed that only 19 missense mutations could cause LCH, which scattered in the LHCGR gene. CONCLUSIONS: The new mutation c.458T> C (p.Leu153Pro) of the LHCGR gene found in the 46, XY DSD patient may cause LCH by interfering with the binding function of the ligand, which has enriched the LHCGR gene mutation database and provided some reference for the studies on the LCH genotype, its phenotypic correlation and gene functions.

[Study on inducing an artemisinin-resistant line of Plasmodium berghei].

OBJECTIVE: To induce a line of Plasmodium berghei with resistance to artemisinin. METHODS: The major methods included blood transmission from passage to passage and progressive increase of drug pressure. RESULTS: The resistant lines were developed by different protocols: (A) The initial dosage of artemisinin was 126.2 mg/kg which was increased by 60 mg/kg for the next passage and boosted by 126.2 mg/kg for every other passage. As developed to passage 60 and 76, the resistant index was 18.39:1 and 14.89:1 respectively, then decreased gradually. For passage 108, the dosage was 8,862.5 mg/kg, but the resistant index was only 10.49:1. (B) Using passage 66 from (A) as the source, a dosage of 4,000 mg/kg was given each week, the resistance of the passage 40 increased significantly with an index of 27.5:1. (C) Using passage 19 of (B) as the source, drug was administered at the dose of 2,000 mg/kg each week. The resistant index of passage 15 was 17.41:1. CONCLUSION: Line of P. berghei with medium level resistance to artemisinin was established.