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In this study, we assessed the therapeutic effects of Macleaya cordata (Willd). R. Br.-derived protopine-type alkaloids (MPTAs) in a mouse model of lipopolysaccharide (LPS)-induced intestinal inflammation. The experimental design involved the allocation of mice into distinct groups, including a control group, a model group treated with 6 mg/kg LPS, a berberine group treated with 50 mg/kg berberine hydrochloride and low-, medium- and high-dose MPTA groups treated with 6, 12 and 24 mg/kg MPTAs, respectively. Histological analysis of the ileum, jejunum and duodenum was performed using Hematoxylin and Eosin (H&E) staining. Moreover, the quantification of intestinal goblet cells (GCs) was performed based on PAS staining. The serum levels of IL-1ß, IL-6, IL-8 and TNF-α were quantified using an enzyme-linked immunosorbent assay (ELISA), while the mRNA levels of TLR4, NF-κB p65, NLRP3, IL-6 and IL-1ß were assessed using quantitative PCR (qPCR). The protein levels of TLR4, Md-2, MyD88, NF-κB p65 and NLRP3 were determined using Western blotting. Furthermore, the 16S rDNA sequences of bacterial taxa were amplified and analysed to determine alterations in the gut microbiota of the mice following MPTA treatment. Different doses of MPTAs were found to elicit distinct therapeutic effects, leading to enhanced intestinal morphology and an increased abundance of intestinal GCs. A significant decrease was noted in the levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α). Additionally, the protein levels of TLR4, MyD88, NLRP3 and p-p65/p65 were markedly reduced by MPTA treatment. Furthermore, 16S rDNA sequencing analysis revealed that the administration of 24 mg/kg MPTAs facilitated the restoration of microbial composition.

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Ulcerative colitis (UC) is an inflammatory bowel disease (IBD), and its pathogenesis is related to intestinal mucosal barrier damage and gut microbiota imbalance. Protopine (PRO), an isoquinoline alkaloid, is one of the main anti-inflammatory ingredients of traditional Chinese medicine Macleaya cordata(Willd.) R. Br. This study investigated the effects of PRO on the intestinal mucosal barrier and gut microbiota in dextran sodium sulfate (DSS)-induced colitis mice. C57BL/6J mice were treated with 3% DSS in drinking water to induce acute colitis, while PRO was administered orally once daily for 7 days. The results showed that PRO administration significantly alleviated the symptoms of DSS-induced colitis in mice and inhibited the expression of inflammation-related genes. In addition, PRO restored the integrity of the intestinal barrier in colitis mice by restoring colonic mucin secretion and promoting the expression of tight junction proteins. Furthermore, PRO alleviated the DSS-induced gut microbiota dysbiosis by decreasing the abundance of Proteobacteria, Escherichia-Shigella and Enterococcus, as well as enhancing the abundance of beneficial bacteria, such as Firmicutes and Akkermansia. These findings suggested that PRO effectively alleviated DSS-induced ulcerative colitis by suppressing the expression of inflammation-related genes, maintaining the intestinal mucosal barrier and regulating the intestinal microbiota.

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Allocryptopine (ALL) is an isoquinoline alkaloid extracted from Macleaya cordata(Willd). R. Br., which has been claimed to have anti-inflammatory and neuroprotection properties. However, the mechanism by which ALL ameliorates inflammatory bowel disease (IBD) remains unclear. Here, we used network pharmacology and quantitative proteomic approaches to investigate the effect of ALL on IBD pathogenesis. Network pharmacology predicted potential targets and signaling pathways of ALL's anti-IBD effects. As predicted by network pharmacology, gene ontology (GO) analysis, in terms of the proteomic results, showed that the immune response in mucosa and antimicrobial humoral response were enriched. Further study revealed that the ALL-related pathways were the chemokine signaling pathway and apoptosis in the Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, we identified AKT1 as a hub for the critical pathways through protein-protein interaction (PPI) network analysis. Similar to mesalazine (MES), Western blot verified that ALL downregulated upstream chemokine CX3CL1 and GNB5 content to reduce phosphorylation of AKT and NF-κB, as well as the degree of apoptosis, to improve inflammatory response in the colon. Our research may shed light on the mechanism by which ALL inhibits the CX3CL1/GNB5/AKT2/NF-κB/apoptosis pathway and improves the intestinal barrier to reduce colitis response and act on the CX3CL1-CX3CR1 axis to achieve neuroprotection.

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Spontaneous unilateral cryptorchid boars have one testis in the abdomen or inguinal canal, causing its temperature to be at or near the body temperature, which impairs spermatogenesis, although the histomorphometry and molecular mechanisms underlying this process remain unclear. The aim of the present study was to determine the histomorphometry, proliferation, apoptosis, and autophagy alterations in spermatogonia and Sertoli cells in unilateral cryptorchid, scrotal (contrascrotal), and preweaning piglet (preweaning) testes. Histomorphometrical analysis of cryptorchid testes showed that the seminiferous tubules contained only Sertoli cells and a few spermatogonia, but did not contain post-meiotic germ cells. The number of spermatogonia markedly decreased, and the number of Sertoli cells did not change remarkably in cryptorchid testes. TUNEL assay results showed that apoptosis signals were predominantly observed in spermatogonia. In cryptorchid and contrascrotal testes, proliferating cell nuclear antigen (PCNA) and LC3 were located in spermatogonia. The number of PCNA-positive, TUNEL-positive, and LC3-positive germ cells was low, and the protein and mRNA levels of PCNA and LC3 were significantly decreased in cryptorchid testes. Taken together, the number of Sertoli cells did not change remarkably, whereas the number of germ cells decreased in the cryptorchid testes, compared with that in the contrascrotal testes. Insufficient proliferation, excessive apoptosis, and autophagy were involved in the regulation of the decrease in spermatogonia in cryptorchid boar testes.

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It was well known that a critical process of oogenesis in the female mammalian was the entry of mitotic oogonia into meiosis. Early studies from model animal mice suggested that the retinoic acid (RA) response signal protein STRA8 (stimulated by retinoic acid gene 8) and the meiosis-specific chromosomal behavior marker protein SCP3 (Synaptonemal Complex Protein 3) were two crucial molecular markers during meiosis. The expression of STRA8 and SCP3 at different stages in rat ovaries was investigated by immunohistochemistry, qRT-PCR and Western Blot. Immunohistochemistry results showed that STRA8 and SCP3 were mainly expressed in embryonic stage. And STRA8 was expressed in the cytoplasm and nucleus of the ovaries after birth. qRT-PCR and Western Blot results showed that the mRNA and protein levels of STRA8 and SCP3 were expressed in embryonic stage. The expression of STRA8 and SCP3 indicated germ cells enter meiosis in rats embryo, and STRA8 and SCP3 could serve as molecular markers for the meiosis in rats. The localization of STRA8 in the nucleus increased the possibility that STRA8 might act as transcription factor or activate transcription to function after birth.

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Body temperature could lead to interruption of spermatogenesis, but the molecular mechanism was still unclear. Cryptorchidism was defined as the failure of testes to enter the scrotum, which exposed the testes to body temperature. Meiosis was a unique feature of germ cell development. Whether cryptorchidism damage the initiation of meiosis in boars had not been reported. The aim of this study was to determine whether spermatogonia in the cryptorchid testes entered into meiosis by detecting meiosis-related markers stimulated by retinoic acid gene 8 (STRA8) and synaptonemal complex protein 3 (SCP3). Three boars with spontaneous unilateral abdominal cryptorchidism were used. The testis located in the abdomen was cryptorchidism group, the scrotal testis of the same animal was used as control. HE results showed that only Sertoli cells, and a few spermatogonia remained in the seminiferous tubules, and no spermatids were seen compared with the control. Immunohistochemistry results showed that in both control and cryptorchidism group, STRA8 was mainly expressed in the nucleus of spermatogonia and spermatocytes. In control group, SCP3 was expressed in the nucleus of spermatocytes. In cryptorchidism group, SCP3 immunopositive cells were also observed. qRT-PCR and Western Blot results showed that the mRNA and protein levels of STRA8 and SCP3 were significantly decreased in cryptorchid boars. The expression of STRA8 and SCP3 in cryptorchidism suggested that spermatogonia could still enter meiosis in cryptorchid boars.

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Heat stress damaged spermatogenesis and semen quality, however, the exact molecular mechanism is not clear. The objective of this study was to determine the effects of elevated ambient temperature and local testicular heating on the expressions of heat shock protein 70 and androgen receptor in boar testes. A growing body of evidence demonstrated that germ cell apoptosis can be aggravated by heat stress and androgen deprivation, and at normal temperature, withdrawal of androgen led to germ cell apoptosis. There were no reports that heat stress damaged spermatogenesis has relationship with androgen. In this study, adult boars (Landrace, n = 9) were used and randomly divided into: control group (CON), 20-27 °C; environmental hyperthermia group (EH), 37-40 °C, 3 h/d 42 d; and local testicular heating group (LTH), 42 °C 1 h. After heat treatments, all boars were castrated and the testes were harvested. qRT-PCR and Western Blot results showed that the mRNA and protein levels of heat shock protein 70 and androgen receptor were significantly increased after heat treatments. Immunohistochemistry results showed that heat stress caused a redistribution of heat shock protein 70 from the cytoplasm to the nucleus, and androgen receptor was mainly expressed in Sertoli cells. These results indicated that heat stress promoted the inhibition of heat shock protein 70 on the androgen receptor, suggesting that the possible mechanism of heat stress damaged spermatogenesis and semen quality was that heat stress reduced the sensitivity of testicular cells to androgen by up-regulating heat shock proteins.

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