Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Background: Excessive growth of keratinocytes is the critical event in the etiology of psoriasis.However, the underlying molecular mechanism of psoriatic keratinocyte hyperproliferation is still unclear. Objective: This study aimed to figure out the potential contributory role of S-phase kinase-associated protein 2 (SKP2) in promoting the hyperproliferation of keratinocytes in psoriasis. Methods: We analyzed microarray data (GSE41662) to investigate the gene expression of SKP2in psoriatic lesion skins compared with their adjacent non-lesional skin. Then, we further confirmed the mRNA and protein expression of SKP2 in human psoriatic skin tissues, imiquimod (IMQ)-induced psoriatic mice back skins and tumor necrosis factor α (TNF-α), interleukin (IL)-17A and IL-6-stimulated keratinocytes by using real-time quantitative polymerase chain reaction and western blot (WB). Furthermore, we explored the potential pathogenic role and its underlying cellular mechanism of SKP2 in promoting keratinocytes hyperproliferation through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle detection, 5-ethynyl-2′-deoxyuridine staining and WB. Finally, we determined whether inhibition of SKP2 can effectively alleviate the keratinocytes hyperproliferation in vivo. Results: We identified that SKP2 is aberrantly upregulated in the psoriatic lesion skin and cytokines-stimulated keratinocytes. Moreover, upregulated SKP2 augments cytokines-induced keratinocytes hyperproliferation. Mechanistically, enhanced SKP2 increased the S phase ratio through inhibiting Cyclin-Dependent Kinase Inhibitor p27 (P27 Kip1) expression. Correspondingly, suppression of SKP2 with SMIP004 can significantly ease the epidermis hyperplasia in vivo. Conclusion Our results suggest that elevated SKP2 can empower keratinocytes proliferation and psoriasis-like epidermis hyperplasia via downregulation of P27 Kip1. Therefore, targeting SKP2-P27 Kip1 axis might be a promising therapeutic strategy for the treatment of psoriasis in future.

Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.

Objective To evaluate the functional and oncological outcomes of patients with locally advanced prostate cancer (PCa) treated by hormone therapy combined with extra-fascia1 laparoscopic radical prostatectomy (LRP) plus extended lymph node dissection (ePLND).Methods From January 2004 to June 2016,a total of 255 PCa cases (pT3-4NxM0) who received LRP plus ePLND were enrolled into our study.The mean age of the patients was 67 (range 44-88) years,and median PSA level was 21.2 (range 0.6-454.0) ng/ml.The patients were divided into earlier group (from January 2004 to December 2011,160 cases) and later group (from January 2012 to June 2016,95 cases) according to different treatment periods.The baseline demographics between the two groups were similar.All patients routinely received adjuvant hormone therapy (AHT) postoperatively.The patients in the later group underwent collapsin response mediator protein 4 (CRMP4) methylation study on the prostatic biopsy preoperatively.Those with a CRMP4 methylation level ＞ 15％ or rectum/bladder neck invasion,were treated by neoadjuvant hormone therapy (NHT) for 3-6 months.Positive surgical margin (PSM),progression-free survival (PFS),cancer-specific survival (CSS),overall survival (OS) and postoperative continence rates between the two groups were analyzed and compared.Results The mean operative time of the earlier and later group were (239 ±65) min and (203±51) min,mean blood loss were (109-±65) ml and (96-±44) ml,mean dissected nodes were (19 ± 5) and (21 ± 7),respectively (all P ＞ 0.05).The total PSM rate was 19.2％,and PSM rates of the two groups were 23.1％ and 12.6％ (P =0.04).All the 255 cases received AHT and 25 cases in the later group underwent NHT.The median follow-up time was 73 months (range 10-152 months).The total 5-year PFS,CSS and OS rates were 77.7％,94.3％ and 87.1％,respectively,and the rates between groups were 73.8％ vs.86.1％ (P=0.03),93.1％ vs.98.6％ (P=0.07),and 85.0％ vs.92.8％ (P =0.11),respectively.The 1-year postoperative continence rates were 91.9％ vs.97.9％ (P =0.09).Conclusions Hormone therapy combined with LRP plus ePLND represents an oncological and functional effective option in patients with locally advanced PCa,and improved PFS might be acquired by preoperative tumor staging.