PI3K/AKT signaling pathway associates with pyroptosis and inflammation in patients with endometriosis.

Endometriosis (EMS) is known to be closely associated with inflammation. We evaluate the possible mechanism linking the PI3K/AKT signaling pathway with pyroptosis and inflammation in EMS. We collected 30 patients undergoing laparoscopic for endometriosis as the EMS group and those undergoing surgery for uterine fibroids as the control group, from whom we collected serum, normal endometrium, eutopic endometrium and ectopic endometrium. Transmission electron microscopy (TEM) was used to observe the internal structure of endometrial cells. Western Blot was used to detect the protein expression of PI3K, P-PI3K, AKT, P-AKT, NLRP3, Caspase-1, GSDMD, and GSDMD-N. Immunohistochemistry (IHC) staining was used to detect the expression of PI3K, AKT, NLRP3, Caspase-1, GSDMD, and GSDMD-N proteins. Immunofluorescence (IF) staining was used to observe the expression of GSDMD-N. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the mRNA levels of PI3K, AKT, NLRP3, Caspase-1, GSDMD, and GSDMD-N. ELISA was used to detect serum levels of IL-1ß, IL-18, TLR4, and NF-κB. We found that activation of PI3K/AKT signaling pathway in endometriosis significantly increased the level of cellular pyroptosis and inflammatory factors. Our results suggest that there is a positive correlation between the PI3K/AKT signaling pathway and pyroptosisas well as inflammation in EMS patients.

Marinobacter panjinensis sp. nov., a moderately halophilic bacterium isolated from sea tidal flat environment.

Two moderately halotolerant bacterium strains, designated PJ-16T and PJ-38, were isolated from a tidal flat of the red beach in Panjin City, Liaoning Province, PR China. Cells were found to be Gram-stain-negative, aerobic, motile, rod-shaped with a single polar flagellum. Optimum growth of strain PJ-16T occurred at 30 °C, pH 7.0 and 0.2-8.0  % (w/v) NaCl, and strain PJ-38 at 30 °C, pH 6.0-7.0 and 0.2-8.0  % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PJ-16T was most closely related to Marinobacter denitrificans KCTC 62941T (99.2 % 16S rRNA gene sequence similarity), Marinobacter algicola DSM 16394T (98.6 %), Marinobacter salarius JCM 19399T (98.4 %) and Marinobacter confluentis KCTC 42705T (98.2 %), and strain PJ-38 was most closely related to M. denitrificans KCTC 62941T (99.1 %), M. algicola DSM 16394T (98.6 %), M. salarius JCM 19399T (98.4 %) and M. confluentis KCTC 42705T (98.1 %). The G+C content of the genomic DNA of strain PJ-16T based on its draft genomic sequence was 57.4 mol%. The major cellular fatty acids of strain PJ-16T were C16 : 0, C16 : 1 ω7c/C16 : 1 ω6c and C18 : 1 ω9c. The major respiratory quinone of PJ-16T was ubiquinone-9 and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The results of the phenotypic, phylogenetic and genomic analyses revealed that strains PJ-16T and PJ-38 represent a novel species of the genus Marinobacter, and the name Marinobacter panjinensis sp. nov. is proposed. The type strain is PJ-16T (= CGMCC 1.13694T= KCTC 72023T).

Bushen Wenyang Huayu Decoction inhibits autophagy by regulating the SIRT1-FoXO-1 pathway in endometriosis rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Wenyang Huayu Decoction (BWHD) is a traditional Chinese medicine for tonifying kidney and warming Yang, thereby resolving blood stasis and relieving pain. BWHD can significantly improve the clinical symptoms of patients with endometriosis (EMs), but its mechanism is still unclear. AIM OF THE STUDY: We evaluated the expression and role of the SIRT1-FoxO-1 pathway and autophagy levels in EMs rats. The therapeutic effects and potential therapeutic mechanisms of BWHD were also investigated. METHODS: Twenty rats were randomized into the sham group and eighty rats were used for model establishment by autologous transplantation. After successful modeling, they were randomized into the model, BWHD, EX527+BWHD and EX527 groups, with 20 rats in each group. All rats were intragastrically administered with for 3 weeks. Localization of Sirtuin 1 (SIRT1), Forkhead boxO-1 (FoXO-1), Beclin-1, autophagy-related 5 (Atg5) and autophagy-related 7 (Atg7) was determined by immunohistochemical staining. The expression of the above proteins was determined by Western blot and their messenger RNA (mRNA) levels were detected by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). RESULTS: The protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in the model group were markedly increased, while that of SIRT1 was markedly decreased relative to the sham group (p < 0.05 and p<0.01, respectively). Results showed that the protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of BWHD group were lower, while SIRT1 expression was higher than in the model group (p < 0.05 and p<0.01, respectively). Furthermore, protein and mRNA expression levels of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of EX527 group were higher, while SIRT1 level was significantly lower than in the model group (p < 0.05 and p < 0.01, respectively). The EX527-induced changes in protein and mRNA expressions were reversed in the EX527+BWHD group (p < 0.05 and p < 0.01, respectively). CONCLUSIONS: BWHD inhibits autophagy by up-regulating SIRT1 and down-regulating FoXO-1 expression in EMs via the SIRT1-FoXO-1 signaling pathway. Therefore, it is a potential treatment for EMs.

Bushen Wenyang Huayu Decoction Targets TLR4/NF-κB Mediated Autophagy to Treat Endometriosis Effectively.

Endometriosis has been found to be closely related to autophagy. This study aimed to elucidate the possible mechanism of Bushen Wenyang Huayu Decoction (BWHD) in treating endometriosis (EMs) by targeting TLR4/NF-κB-mediated autophagy. Autologous grafting was used to generate the EMs model in rats. Once the model was developed, BWHD high-dose and low-dose groups received intragastric administration of BWHD, and the gestrinone group served as a positive control. Immunofluorescence labeling and Western blotting were used for the protein expression of toll-like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), Beclin-1, and selective autophagy connector protein P62 (P62). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze mRNA levels of TLR4, NF-κB, Beclin-1, and P62. We found that BWHD significantly reduced the size of ectopic lesions in rats with EMs, regulated reproductive hormone levels, and alleviated the cell autophagy level. It suggested that BWHD could be an effective treatment of EMs by targeting TLR4/NF-κB signaling pathway.

Involvement of bradykinin and bradykinin B1 receptor in patients with endometriosis.

Endometriosis (EM), a benign aseptic inflammatory disease, is associated with the presence of endometrial foci. Pain, one of its typical symptoms, has been reported as a constant stressor, but the etiology and pathogenesis of EM-associated pain are unclear. In the present study, eutopic and ectopic endometrium samples from women with EM (n=50) and normal endometrium samples from control subjects (n=20) were collected. Serum levels of prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α) and bradykinin (BK) were measured using commercial ELISA kits. The expression of the BKB1 receptor (BKB1R) protein was evaluated by immunohistochemical staining and western blot assay. The mRNA expression of BKB1R was measured by reverse transcription-quantitative PCR. The results revealed that there was a substantial increase in the protein and mRNA expression of BKB1R, as well as the release of PGE2, PGF2α and BK in the blood, in the EM group compared with that in the control group. Moreover, PGE2, PGF2α and BK levels were significantly correlated with each other, as well as with the pain intensity of EM. The increased expression levels of BKB1R protein and mRNA were positively correlated with the pain degree of EM. Thus, these data indicated that BK and BKB1R were involved in the pathological onset of EM-associated pain and that they may play an important role in EM-related pain by inducing PGE2 and PGF2α. The data indicate a potential new therapeutic target for EM-related pain.

Sphingobacterium puteale sp. nov., isolated from a deep subsurface aquifer.

A bacterial strain M05W1-28T was isolated from a well that collected water for irrigation from a deep aquifer at a depth of 400 m. Cells were observed to be rod-shaped, non-motile, aerobic, stained Gram-negative. Optimal growth was obtained at pH 7.0 (range: 6.0-9.0), 28 °C (range: 15-37 °C) and 0 % NaCl (range: 0-1.5 %, w/v) in modified tryptic soy broth (mTSB) without added NaCl and R2A. The cells were found to be positive for catalase and oxidase activities. The major fatty acids (>10 %) were identified as summed feature 3 (C16 : 1 ω7c / C16 : 1 ω6c) and iso-C15 : 0. The major polar lipids were phosphatidylethanolamine, glycolipid, phosphoglycolipids, phospholipids, and unidentified lipids. The major respiratory quinone was menaquinone-7 (MK-7). The genomic G+C content of strain M05W1-28T was 40.7 %. Based on similarities of 16S rRNA gene sequences, strain M05W1-28T was affiliated with the genus Sphingobacterium, exhibiting the highest sequence similarities with S. multivorum LMG 8342T (97.5 %), S. ginsenosidimutans THG07T (97.1 %) and less than 97.0 % to other members of the genus. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation values (dDDH) between M05W1-28T and S. multivorum LMG 8342T were 78.1 and 22.5 %, respectively. Phenotypic characteristics including enzyme activities and carbon source utilisation differentiated the strain from other Sphingobacterium species. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain M05W1-28T represented a novel species within the genus Sphingobacterium, for which the name Sphingobacterium puteale sp. nov. is proposed. The type strain is M05W1-28T (=CGMCC 1.13711T=KCTC 72027T).

Sandarakinorhabdus cyanobacteriorum sp. nov., a novel bacterium isolated from cyanobacterial aggregates in a eutrophic lake.

A bacterial strain, designated TH057T, was isolated from cyanobacterial aggregates in a eutrophic lake in China. Cells were observed to be slightly curved, rod-shaped, capsule-forming and stained Gram-negative. Optimal growth was obtained at pH 7.0 (range: pH 5-9) and 30 °C (range: 20-37 °C) in R2A broth. According to the absorption spectrum, carotenoids (455 and 490 nm) and light-harvesting complex LHI (857 nm) were present in the cells. The cells were found to be positive for oxidase and catalase activities. The major respiratory quinone was ubiquinone Q-10. The major fatty acids were identified as C17 : 1ω6c, C16 : 1ω7c/C16 : 1ω6c, C18 : 1ω6c/C18 : 1ω7c and C16 : 0. The major polar lipids were found to consist of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, unidentified glycolipid and two sphingoglycolipids. Strain TH057T shared highest 16S rRNA gene sequence similarity to Sandarakinorhabdus limnophila so42T (96.8 %), followed by Polymorphobacter fuscus D40PT (95.8 %). The genomic G+C content of strain TH057T was 66.1 mol% based on total genome calculations. The average nucleotide identity and the digital DNA-DNA hybridization value for the complete genomes were 81.0 and 23.0 % between strain TH057T and Sandarakinorhabdus limnophila so42T. The phenotypic, chemotaxonomic and phylogenetic properties, and genome analysis suggested that strain TH057T represents a novel species within the genus Sandarakinorhabdus, for which the name Sandarakinorhabduscyanobacteriorum sp. nov. is proposed. The type strain is TH057T (=CGMCC 1.15803T=LMG 30294T).

Chryseobacterium zhengzhouense sp. nov., isolated from groundwater of the well in a vegetable field, and emended description of the genus Chryseobacterium.

A Gram-negative, strictly aerobic, non-motile, asporogenous rod-shaped bacterium, designated M05W1-39A1(T), was isolated from a Chinese cabbage farmland located in Zhengzhou. China, and subjected to a taxonomic study. Strain M05W1-39A1(T) was found to grow optimally at 25-30 °C, at pH 6.0-7.0 and in the presence of 0.5-2.0 % (w/v) NaCl. According to phylogenetic analysis using 16S rRNA gene sequences, strain M05W1-39A1(T) belongs to the genus Chryseobacterium and is closely related to Chryseobacterium arachidis LMG 27813(T) (98.8 %) and Chryseobacterium geocarposphaera LMG 27811(T) (98.1 %). The DNA G + C content was determined to be 35.3 mol%. The respiratory quinone was identified as MK-6 and the predominant cellular fatty acids as iso-C15:0, Summed feature 3 (C16:1 ω7c/C16:1 ω6c), iso-C17:0 3-OH and Summed feature 9 (iso-C17:1 ω9c). Based on the genotypic, chemotaxonomic and phenotypic data, strain M05W1-39A1(T) is concluded to represent a novel species of the genus Chryseobacterium, for which the name Chryseobacterium zhengzhouense sp. nov. is proposed. The type strain is M05W1-39A1(T) (=HNMC11208(T) = CGMCC 1.15067(T) = JCM 30863(T)).

Methylopila henanense sp. nov., a novel methylotrophic bacterium isolated from tribenuron methyl-contaminated wheat soil.

A bacterial strain, designated LYBFD3-16A2(T), was isolated from tribenuron methyl contaminated wheat soil. Cells were observed to be Gram-negative short rods with a single flagellum. The strain was found to utilize methanol, glucose, maltose and mannitol as carbon and energy sources, and utilized glutamate, leucine, phenylalanine as organic nitrogen sources. Strain LYBFD3-16A2(T) was found to be aerobic, to form urease, produce hydrogen sulfide and reduce nitrate to nitrite. The indole test in tryptone broth was observed to be positive. The major cellular fatty acids were identified as C18:1ω7c (81.3 %), 11-methylC18:1ω7c (7.9 %), C18:0 (3.0 %) and C16:0 (3.0 %). The major phospholipids were identified as phosphatidylcholine, phosphatidylethanolamine, phosphatidyglycerol and diphosphatidylglycerol. The main ubiquinone was identified as Q-10. The DNA G+C content was determined to be between 70.2 and 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated the affiliation of strain LYBFD3-16A2 to members of the genus Methylopila. The DNA-DNA hybridization values of the novel strain with the type strains of the most closely related species Methylopila musalis MUSA(T) and Methylopila jiangsuensis JZL-4(T) were 35.4 % and 31.4 % respectively. The genotypic and phenotypic characterization, along with chemotaxonomic properties of strain LYBFD3-16A2(T), showed that the strain represents a novel species of the genus Methylopila for which the name Methylopila henanense sp. nov. is proposed. The type strain is LYBFD3-16A2(T) (=CGMCC1.10703(T) = LMG 25959(T)).

Pseudomonas nitritireducens sp. nov., a nitrite reduction bacterium isolated from wheat soil.

A Gram-negative, non-mobile, polar single flagellum, rod-shaped bacterium WZBFD3-5A2(T) was isolated from a wheat soil subjected to herbicides for several years. Cells of strain WZBFD3-5A2(T) grow optimally on Luria-Bertani agar medium at 30 °C in the presence of 0-4.0 % (w/v) NaCl and pH 8.0. 16S rRNA gene sequence analysis revealed that strain WZBFD3-5A2(T) belongs to the genus Pseudomonas. Physiological and biochemical tests supported the phylogenetic affiliation. Strain WZBFD3-5A2(T) is closely related to Pseudomonas nitroreducens IAM1439(T), sharing 99.7 % sequence similarity. DNA-DNA hybridization experiments between the two strains showed only moderate reassociation similarity (33.92 ± 1.0 %). The DNA G+C content is 62.0 mol%. The predominant respiratory quinine is Q-9. The major cellular fatty acids present are C(16:0) (28.55 %), C(16:1ω6c) or C(16:1ω7c) (20.94 %), C(18:1ω7c) (17.21 %) and C(18:0) (13.73 %). The isolate is distinguishable from other related members of the genus Pseudomonas on the basis of phenotypic and biochemical characteristics. From the genotypic, chemotaxonomic and phenotypic data, it is evident that strain WZBFD3-5A2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitritereducens sp. nov. is proposed. The type strain is WZBFD3-5A2(T) (=CGMCC 1.10702(T) = LMG 25966(T)).