ABSTRACT
ObjectiveTo observe the effect of acupuncture “Zhibian （BL 54）-to-Shuidao （ST 28）” on the reproductive function of asthenospermia model mice and to explore the possible mechanism. MethodsForty-two male C57BL/6 mice were randomly divided into blank group， model group， and acupuncture group， with 14 mice in each group. Cyclophosphamide 30 mg/（kg·d） was injected intraperitoneally for 7 days to establish model of asthenospermia for the model group and the acupuncture group， while 0.9% sodium chloride solution 10 ml/（kg·d） was injected intraperitoneally for 7 days in the blank group. After successful modeling， mice in the acupuncture group received acupuncture at “Zhibian （BL 54）-to-Shuidao （ST 28）” once a day for 20 minutes， 6 times a week for 3 weeks； the remaining two groups were fixed without acupuncture. Daily observations were made on the general conditions of mice in all groups， and changes in body weight after intervention were recorded. On the next day after completing the treatment， 6 male mice selected randomly from each group to test the sperm quality as well as testicular and epididymal weights， and calculate the corresponding indices； ELISA was used to test the levels of testosterone （T）， follicle-stimulating hormone （FSH）， and luteinizing hormone （LH） in serum； HE staining and TUNEL staining were performed to observe the pathological morphology and apoptosis of testicular and epididymal tissues； fluorescence quantitative PCR and western blot were used to detect changes in the expression of apoptosis-related factors （Fas）， Fas-associated death domain protein （FADD）， B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cysteine-aspartic acid protease （caspase）-3 mRNA and protein in testicular tissue. The remaining 8 male mice in each group were housed with estrus-cycling mice of the same strain at 1∶1 ratio， and the pregnancy rate and number of embryos per litter in each group were determined after mating. ResultsIn comparison with the blank group， mice in the model group exhibited reduced body weight， decreased testicular mass， testicular index， epididymal mass， and epididymal index. Additionally， there was a decrease in total sperm count， sperm motility， and sperm viability. Serum levels of T， FSH， and LH were reduced. The apoptosis rate increased， and the expression levels of Fas， FADD， Bax， and Caspase-3 mRNA and proteins were elevated， while Bcl-2 mRNA and protein expression levels decreased （P＜0.01）. Pathological abnormalities were observed in testicular and epididymal tissues， with disrupted arrangement of seminiferous tubules and a decreased number of spermatogenic cells within the tubular lumen. Furthermore， the pregnancy rate and the number of embryos per litter decreased significantly after mating （P＜0.01）. In comparison with the model group， mice in the acupuncture group showed improvements in testicular mass， testicular index， epididymal mass， epididymal index， total sperm count， sperm motility， and sperm viability. Serum levels of T， FSH， and LH increased. The apoptosis rate decreased， and the expression levels of Fas， FADD， Bax， and Caspase-3 mRNA and proteins decreased， while Bcl-2 mRNA and protein expression levels increased （P＜0.05 or P＜0.01）. Morphological improvements were observed in testicular and epididymal tissues， with a compact arrangement of seminiferous tubules and an increased number of spermatogenic cells within the tubular lumen. Furthermore， the pregnancy rate and the number of embryos per litter increased significantly after mating （P＜0.01）. ConclusionAcupuncture “Zhibian （BL 54）-to-Shuidao （ST 28）” can improve testicular tissue apoptosis and enhance reproductive function in a mouse model of asthenospermia. Its mechanism may be associated with the modulation of key factors in the extrinsic membrane receptor pathway （Fas-mediated pathway） and the intrinsic mitochondrial pathway （Bcl-2/Bax-regulated pathway） in testicular tissue.
ABSTRACT
ObjectiveTo investigate the changes and potential effects of differentially expressed microRNAs (miRNAs) in the development and progression of liver injury in a mouse model of autoimmune hepatitis (AIH) induced by concanavalin A (ConA). MethodsEight healthy male specific pathogen-free C57BL/6 mice were randomly divided into model group and control group, with four mice in each group. The mice in the model group were given tail vein injection of ConA 15 mg/kg, and those in the control group were given an equal volume of normal saline. All mice were sacrificed after 8 hours of modeling, Total RNA in liver tissue was extracted, gene microarray was used to screen out differentially expressed miRNAs, and target prediction and function analysis were performed for upregulated and downregulated miRNAs. The independent samples t-test was used for comparison of differentially expressed miRNAs between two groups. ResultsThe principal component analysis showed that the inter-group difference of the data extracted by gene microarray met the conditions for further analysis. Compared with the control group, the model group had 31 upregulated miRNAs and 18 downregulated miRNAs in mouse liver, which had a regulatory relationship with 959 target genes (601 upregulated genes and 358 downregulated genes). GO analysis showed that in the model group, the target genes of the upregulated miRNAs mainly had the molecular functions such as “DNA binding” (P=1.47×10-6), participated in the biological processes such as “transcription, DNA-templated” (P=2.36×10-7), and were mainly enriched in the cellular components such as “neuronal cell body” (P=5.99×10-6), while the target genes of the downregulated miRNAs had the molecular functions such as “RNA polymerase II proximal promoter sequence-specific DNA binding” (P=2.49×10-6), participated in the biological processes such as “regulation of transcription, DNA-templated” (P=1.64×10-11), and were mainly enriched in the cellular components such as “nucleoplasm” (P=4.30×10-10). KEGG pathway enrichment analysis showed that the target genes of the upregulated miRNAs were mainly enriched in “Endocytosis” (P=0.000 4), while the target genes of the downregulated miRNAs were mainly enriched in the “Hippo signaling pathway” (P=0.004), and the above functional analysis results were statistically significant (P＜0.05). ConclusionThere are differentially expressed miRNAs in the pathogenesis of AIH, and these differentially expressed miRNAs can provide new targets for the clinical treatment of AIH.