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ObjectiveTo investigate the effect of modified Weijingtang on the pyroptosis of RAW264.7 macrophages via the cysteinyl aspartate-specific protease-1 （Caspase-1）/gasdermin D （GSDMD） pathway. MethodLipopolysaccharide （LPS） was used to induce pyroptosis of RAW264.7 cells. The blank group was treated with the blank serum， and the intervention groups were treated with the sera containing different doses of modified Weijingtang. After 24 h， the viability of cells in different groups was examined by the cell counting kit-8 （CCK-8）. The pyroptosis and morphology of cells in each group were observed by a scanning electron microscope and a phase-contrast microscope， respectively. The mRNA and protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）， Caspase-1， and GSDMD in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. The levels of interleukin （IL）-18 and IL-1β in each group were measured by enzyme-linked immunosorbent assay. ResultUnder the electron microscope， RAW264.7 cells presented the best morphology and structure in the blank group and obvious pyroptosis and leakage of cell contents in the model （LPS） group. Compared with the model group， the intervention groups showed reduced pyroptosis to varying degrees， and the high-dose group had the closest cell morphology and structure to the blank group. Under the optical microscope， RAW264.7 cells were spherical in the blank group and irregular with protrusions in the model group. Compared with the model group， the intervention groups showed improved cell morphology， and the cell morphology in the group with the dose of 20% was the closest to that in the blank group. The mRNA and protein levels of NLRP3， Caspase-1， and GSDMD in the model group were higher than those in the blank group （P<0.05）. Compared with the model group， each intervention group showed down-regulated expression of the above indicators （P<0.05）. Compared with the blank group， the model group presented elevated levels of IL-18 and IL-1β （P<0.05）， which were lowered in the intervention （10%， 20%） groups （P<0.01）. ConclusionModified Weijingtang inhibits the pyroptosis of macrophages by down-regulating the Caspase-1/GSDMD pathway and reducing the release of proinflammatory cytokines.
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Objective To investigate the effect of inhibiting lipid synthesis on human skeletal rhabdomyosarcoma cells and its molecular mechanism.Methods The mRNA expression levels of lipid synthesis related genes sterol regulatory element binding protein 1(SREBP1)and squalene cyclooxygenase(SQLE)in human skeletal muscle cells(HSMC)and human skeletal rhabdomyosarcoma cells were detected by tumor gene expression database and verified by qRT-PCR.The concentration of methyl-β-cyclodextrin(mβCD)was determined by cell proliferation as-say.The control group consisted of three human skeletal rhabdomyosarcoma cell lines(RD,SJCRH30,A673),while the experimental group comprised three human skeletal rhabdomyosarcoma cell lines treated with 1mmol/L mβCD.Plate clone formation assay,soft agar,colony formation assay,cell migration assay and cell invasion assay,and tumor formation in nude mice employed to assess changes in proliferation,migration,invasion,and tumor growth of human skeletal rhabdomyosarcoma cells between the control group and mβCD treatment group.The mo-lecular mechanism of mβCD inhibiting the malignancy of human skeletal rhabdomyosarcoma cells was explored by lipoomics and triglyceride(TG)detection.Results Compared with HSMC,the expressions of lipid synthesis relat-ed genes SREBP1 and SQLE significantly increased in human skeleton rhabdomyosarcoma cells(P＜0.001).Compared with the control group,the proliferation,plate cloning,migration,invasion and colony formation ability of human skeleton rhabdomyosarcoma cells in the mβCD group significantly decreased(P＜0.05).The growth of tumor volume and weight in nude mice was also significantly reduced(P＜0.05).The lipidomics results and TG kit analysis revealed a significant reduction in TG content in the mβCD group compared to the control group(P＜0.01).Conclusion m βCD may inhibit the malignant biological behaviors such as proliferation,migration and in-vasion of human skeleton rhabdomyosarcoma cells by reducing TG and other lipid metabolism.
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Objective @# To investigate the effect of SHP2 protein phase separation induced by activation mutation on cell proliferation and its mechanism through construct a mouse model of SHP2E76K mutation . @*Methods @#Hybrid PTPN11 E76K⁃NEO/ + C57BL/6J mouse were hybridizedwith Mx1 ⁃cre tool mice to obtain the required Mx1 ⁃cre;Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + . The later genotype mice were injected with pI⁃pC to induce the expression of Cre enzyme and mutate Ptpn11E76K in bone marrow mesenchymal stem cells(MSC) . The Mx1 ⁃cre ; Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + genotype mice ′s cells were isolated and cultured in vitro and identified as MSCs by immunofluorescence staining . With Ptpn11 + / + MSC as the control group and Ptpn11 E76K/ + MSC as the experimental group , the two kinds of cells were divided into 6 groups by adding drugs : Ptpn11 + / + group ; Ptpn11 + / + + SHP099 group ; Ptpn11 + / + + ET070 group ; Ptpn11 E76K/ + group ; Ptpn11 E76K/ + + SHP099 group ; Ptpn11 E76K/ + + ET070 group . The differences of SHP2 protein phase separation in the six groups were observed by immunofluorescencestaining, and the differences of SHP2 protein expression were detectedby Western blot . CCK⁃8 was used to observe the changes of cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ p ⁃ERK , AMPK/p⁃AMPK , mTOR/p⁃mTOR and other molecules between the six groups . @*Results @# Genotypes Mx1 ⁃cre ; Ptpn11 E76K/ + and Mx1 ⁃cre ; Ptpn11 + / + mice were obtained by genotyping , and the primary MSCs were isolated . Compared with Ptpn11 + / + group , SHP2 proteins in the Ptpn11 E76K/ + group produced more phase separation condensates , and compared with Ptpn11 E76K/ + group , the SHP2 proteins in the Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ ++ ET070 groups produced less phase separation condensates . No difference in SHP2 protein expression levels between groups was detected by Western blot . Compared with Ptpn11 + / + group , the proliferation ability of MSC in Ptpn11 + / + + SHP099 and Ptpn11 + / + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃mTOR decreased, and the expression of p ⁃AMPK protein increased . Compared with Ptpn11 E76K/ + group , the proliferation ability of MSC in Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃ mTOR decreased , and the expression of p⁃AMPK protein increased . @*Conclusion @# SHP2 phase isolation is involved in the alteration of proliferative capacity of SHP2E76K ⁃activated MSCs by stimulating the expression of AMPK⁃mTOR signaling pathway .
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To study the correlation between obesity and breast cancer incidence and progression in postmenopausal female, and provide theoretical instruction for breast cancer prevention and treatment, through being based on literature retrieval platforms such as Pubmed and CNKI, integrating multiple domestic and foreign related documents and various studies, it is found that obesity is one of the most important risk factors for breast cancer incidence and progression in postmenopausal women. On one hand, elevated postmenopausal estrogen levels in obese people directly promote the tumor behavior of breast cancer cells; on the other hand, adipose tissue privides a convenient immune environment for tumor growth by releasing inflammatory mediators, which indirectly promotes tumor development. In addition, obesity also induces the differentiation of adipose stem cell (ASCs) into cancer-associated fibroblasts (CAFs) and enhances the proliferation and invasive potential of breast cancer cells. As mentioned above, obesity increases the risk of breast cancer tumorigenesis and metastasis in postmenopausal women, and increases the risk of cancer-related death in breast cancer patients. The review elaborates the correlation between obesity and breast cancer incidence and progression in postmenopausal women, to provide new ideas for the prevention and treatment of breast cancer, which has essential clinical research value.
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AIM:To explore the regulatory effect of microRNA-3666 (miR-3666) on the expression of its tar-get gene phosphatase and tensin homologue deleted on chromosome ten (PTEN) in leukemic cells.METHODS: miR-3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR.miR-3666 targeting PTEN 3'-untranslated region (3'UTR) was predicted by TargetScan software .3'UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK 2.The reporter activity was evaluated by the Dual-Lu-ciferase Reporter Assay System after the luciferase promoter vector and miRNA were co -transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor ( anti-miR-3666) or a synthetic control miRNA ( anti-miR-C) .The expression of PTEN protein in the above transfected K 562 cells was determined by Western blotting .RESULTS:miR-3666 was up-regulated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells .The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666.Inhi-bition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K 562 cells.CONCLUSION:miR-3666 is over-expressed in leukemic cells .The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN .
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Objective To observe the effects of soy isoflavones (SI) on NO content and NOS activity in myocardial tissues of diabetic rats. Methods Forty-eight SD rats were randomly divided into six groups: normal control group (NC), diabetic control group (DC), SI groups in low, moderate and high doses respectively (L-SI, M-SI, H-SI) and nilestriol group (NI). Except the NC group, the rats were given streptozotocin (STZ) 55mg?kg-1 intraperitneally (ip). From the 7th week, SI in the dosage of 30, 60, 120 mg?kg-1?d-1 was respectively given to L-SI, M-SI, H-SI groups by gastric gavage, nilestriol 0.2 mg?kg-1?per week was given to NI group,and 0.5%CMC-Na10 ml?kg-1?d-1 was given to NC and DC groups. After treatment, fasting blood sugar level, body weight, serum lactate dehydrogenase (LDH)and creatine kinase (CK) contents, and NOS activities and NO content in myocardial homogenate were measured. Results 1) LDH and CK contents in serum were significantly higher in DC group than those in NC group. Compared with DC group, LDH and CK were significantly decreased in M-SI and H-SI groups (P
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AIM: To study a novel gene that probably related with liver regeneration, which was found by representational difference analysis(RDA). METHODS: cDNA sequence, tissue distribution and functions of the novel gene were studied by slot blot, Northern blot, RT-PCR, cDNA library screening and sequence analyzing. RESULTS: Two full-length clones were isolated from cDNA library of rat fetal livers and the sequence analysis identified that the positive cDNA encoded 76 amino acids only; Using the cDNA as a probe, the novel gene showed a specific liver distribution, a moment increasing expression in one hour after partial hepatectomy (PH) and high expression in fetal liver or liver tumor by Northern blot; EGF quickly induced its high expression in primary culture rat hepatocytes(FCS free).CONCLUSION: These results show that the novel gene is an early phase response gene that is closely related to a liver regeneration adjustment. It may encode peptide or has longer sequence at N tip.