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ObjectiveTo explore the possible mechanism of the Yiqi Jiedu formula （YQ） in treating ischemic stroke （IS） from the perspective of the microbial-gut-brain axis （MGBA）. MethodRats were randomly divided into five groups， with six in each group， including sham surgery group， model group， and low， medium， and high dose YQ groups （1， 5， and 25 mg·kg-1）. Except for the sham surgery group， all other groups were established with a middle cerebral artery occlusion （MCAO） model using the thread occlusion method. The success of modeling was determined through neurobehavioral scoring， and the protective effect of YQ on IS was evaluated. Then， the changes in gut microbiota before and after MCAO modeling and YQ administration were compared using 16S rDNA sequencing technology， and the possible biological pathways related to the effect of this formula were analyzed. The expression of inflammatory factors such as interleukin-6 （IL-6）， interleukin-17A （IL-17A）， and interleukin-10 （IL-10） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression of tight junction proteins ZO-1 and Occludin in brain and intestinal tissue， and hematoxylin-eosin staining （HE） was used to observe pathological changes in the cerebral cortex and colon， so as to validate the possible mechanism of action. ResultYQ significantly improved the neurobehavioral score of MCAO rats （P<0.01） and played a good regulatory role in intestinal microbial disorders caused by enriched pathogens and opportunistic pathogens during the acute phase. Among them， significantly changed microorganisms include Morgentia， Escherichia Shigella， Adlercreutzia， and Androbacter. Bioinformatics analysis found that these bacteria may be related to the regulation of inflammation in the brain. Compared with the blank group， the detection of inflammatory factors in the serum of IS model rats showed an increase in inflammatory factors IL-6 and IL-17A （P<0.01） and a decrease in the content of anti-inflammatory factor IL-10 （P<0.01）. Compared with the model group， the content of inflammatory factors IL-6 and IL-17A in the serum of the treatment group decreased （P<0.05）， and that of anti-inflammatory factor IL-10 increased （P<0.01）. The expression results of barrier proteins ZO-1 and Occludin in brain and intestinal tissue showed that the expression levels of both decreased in IS model rats （P<0.05）， while the expression levels of both increased in the treatment group （P<0.05）. ConclusionAcute cerebral ischemia can lead to an imbalance of intestinal microbiota and damage to the intestinal barrier， and it can increase intestinal permeability. YQ can regulate intestinal microbiota imbalance caused by ischemia， inhibit systemic inflammatory response， and improve the disruption of the gut-blood brain barrier， preventing secondary cascade damage to brain tissue caused by inflammation. The MGBA may be an important mechanism against the IS.
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Objective:To investigate differences in clinical characteristics between bullous pemphigoid (BP) patients with stroke and those without, and their relationship with the prognosis of stroke.Methods:A retrospective analysis was performed on medical records of 330 BP inpatients in the First Affiliated Hospital of Zhengzhou University from September 2012 to April 2020. These patients were divided into BP + stroke (ST) group and BP - ST group according to whether they were accompanied by stroke, and clinical manifestations and relevant laboratory examination results were compared between the two groups. According to the stroke outcome score assessed by modified Rankin Scale (mRS), patients in the BP + ST group were further divided into good-prognosis ST group (mRS ≤ 2 points) and poor-prognosis ST group (mRS > 2 points), and subgroup analysis was conducted. Correlations between measurement data (such as age, disease course and laboratory examination results) and mRS scores were analyzed.Results:In the BP - ST group (256 cases), 151 were males and 105 were females, and their age ranged from 19 to 92 (66.8 ± 13.6) years; in the BP + ST group (74 cases), 45 were males and 29 were females, and their age ranged from 48 to 92 (74.6 ± 9.6) years; Compared with the BP - ST group, the BP + ST group showed older age ( t = -5.57, P < 0.001), shorter disease course of BP ( Z = -3.07, P = 0.002), and higher anti-BP180 IgG antibody levels (215.0 [157.2, 283.1] U/ml vs. 155.0 [63.9, 279.8] U/ml; Z = -2.12, P = 0.034). The distribution of skin lesions significantly differed between the two groups ( χ2 = 10.51, P = 0.015), and the BP + ST group showed a significantly lower proportion of patients with generalized lesions ( P<0.05), but a higher proportion of patients with lesions on the limbs ( P<0.05). Subgroup analysis showed significant differences in the patients′ age, BP course, lesion distribution and anti-BP180 IgG antibody levels among the good-prognosis ST group, poor-prognosis ST group and BP - ST group ( F = 10.83, P<0.001; Z = 17.24, P<0.001; χ2 = 15.57, P = 0.026; Z = 6.29, P = 0.043, respectively). There was no significant difference in the age between the good-prognosis ST group and poor-prognosis ST group (adjusted P = 1.000), but the patients were significantly older in the two above groups than in the BP - ST group (adjusted P = 0.001, 0.007, respectively) ; the poor-prognosis ST group showed significantly shorter BP courses (adjusted P = 0.016, < 0.001, respectively) and a higher proportion of patients with lesions on the limbs (both P < 0.05) compared with the good-prognosis ST group and BP - ST group, and significantly higher serum anti-BP180 IgG antibody levels compared with the BP - ST group (226.2 [163.6, 285.8] U/ml vs. 155.0 [63.9, 279.8] U/ml; adjusted P = 0.037). There were no significant differences in the gender distribution, lesional morphology, percentages and counts of peripheral blood eosinophils, serum total IgE levels, and anti-BP230 IgG antibody levels between the BP + ST group and BP - ST group (all P > 0.05), or among the good-prognosis ST group, poor-prognosis ST group and BP - ST group (all P > 0.05). Correlation analysis in the BP + ST group showed a significantly negative correlation between the BP course and mRS scores ( r = -0.33, P = 0.004), and a significantly positive correlation between the anti-BP180 IgG antibody levels and mRS scores ( r = 0.34, P = 0.032) . Conclusion:There were differences in the patients′ age, BP course, lesion distribution, and anti-BP180 IgG antibody levels between the BP patients with stroke and those without, and the differences were more obvious between the poor-prognosis ST group and BP - ST group.
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Objective:To investigate effects of long non-coding growth stasis specific protein 6 antisense RNA1 (lncRNA DLX6-AS1) on the proliferation, migration and invasion of a cutaneous squamous cell carcinoma cell line A431, and to explore the underlying mechanisms.Methods:A dual-luciferase reporter system was used to verify the targeting relationship between lncRNA DLX6-AS1 and miR-16-5p, as well as between miR-16-5p and nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) mRNA. Cultured A431 cells were divided into several groups: si-DLX6-AS1 group and DLX6-AS1-NC group transfected with lncRNA DLX6-AS1 inhibitor and its negative control respectively; anti-miR-16-5p group and anti-miR-NC group transfected with miR-16-5p inhibitor and its negative control respectively; si-NUCKS1 group and NUCKS1-NC group transfected with NUCKS1 inhibitor and its negative control respectively; si-DLX6-AS1+ anti-miR-16-5p group transfected with lncRNA DLX6-AS1 inhibitor followed by miR-16-5p inhibitor, and si-DLX6-AS1+ anti-miR-NC group transfected with lncRNA DLX6-AS1 inhibitor followed by anti-miR-NC; si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1 inhibitor, and si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1-NC. After the above treatment, real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure the mRNA expression of lncRNA DLX6-AS1, miR-16-5p and NUCKS1 in A431 cells, Western blot analysis to determine the protein expression of NUCKS1, Cyclin D1 antibody, matrix metalloproteinase (MMP) 2 and MMP9, cell counting kit-8 (CCK8) assay to detect cell survival rate, and Transwell assay to evaluate cell migratory and invasive abilities. Two-independent-sample t test was used for comparisons between two groups. Results:Dual-luciferase reporter assay showed targeted binding of lncRNA DLX6-AS1 to miR-16-5p, as well as of miR-16-5p to NUCKS1. Compared with the DLX6-AS1-NC group, the si-DLX6-AS1 group showed significantly increased miR-16-5p expression in A431 cells (3.01 ± 0.31 vs. 1.02 ± 0.10, t = 18.33, P < 0.001) , but significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . Compared with the NUCKS1-NC group, the si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression, the si-DLX6-AS1+ anti-miR-16-5p group showed significantly decreased miR-16-5p expression in A431 cells (0.34 ± 0.04) compared with the si-DLX6-AS1+ anti-miR-NC group (1.00 ± 0.12, t = 15.65, P < 0.05) , but significantly increased protein expression of Cyclin D1, MMP2 and MMP9, cell survival rate and numbers of migratory and invasive cells compared with the si-DLX6-AS1+ anti-miR-NC group (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression and knockdown of miR-16-5p, the si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells, as well as cell survival rate and numbers of migratory and invasive cells, compared with the si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group (all P < 0.05) . Conclusion:lncRNA DLX6-AS1 can regulate the proliferation, migration and invasion of A431 cells by targeting miR-16-5p/NUCKS1, suggesting that lncRNA DLX6-AS1 may be a potential molecular target for the treatment of cutaneous squamous cell carcinoma.
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Objective:To evaluate the effects of long non-coding RNA (lncRNA) LEF1-AS1 on proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, and to explore their mechanisms.Methods:Cutaneous squamous cell carcinoma SCC13 cells were divided into si-LEF1-AS1 group transfected with lncRNA LEF1-AS1 interference oligonucleotides (si-LEF1-AS1) , si-NC group transfected with lncRNA LEF1-AS1 nonsense oligonucleotides (si-NC) , miR-612 group transfected with miR-612-overexpressing oligonucleotides, miR-NC group transfected with miR-612 nonsense oligonucleotides (miR-NC) , si-LEF1-AS1+anti-miR-612 group transfected with si-LEF1-AS1 and oligonucleotides against miR-612, and si-LEF1-AS1+anti-miR-NC group transfected with si-LEF1-AS1 and miR-612 nonsense oligonucleotides. Quantitative reverse transcription (qRT) -PCR was performed to determine the relative expression of miR-612 in SCC13 cells, cell counting kit-8 (CCK8) assay to evaluate cellular proliferative activity, flow cytometry to detect cell apoptosis, Transwell assay to assess migratory and invasive abilities of SCC13 cells, and Western blot analysis to determine protein expression of cyclin-dependent kinase 1 (cyclinD1) , cyclinD1 inhibitor p21, Bcl-2 family protein (Bcl-2) , Bcl-2 related X protein (Bax) , matrix metalloproteinase 2 (MMP-2) and MMP-9. The online bioinformatics database LncBase predicted v.2 was employed to predict the complementary sequence between lncRNA LEF1-AS1 and miR-612, and luciferase reporter gene plasmids were constructed by using the complementary/non-complementary sequence, which were co-transfected with miR-612-overexpressing oligonucleotides (miR-612 overexpression group) or miR-NC (overexpression control group) into SCC13 cells in order to verify the binding ability of lncRNA LEF1-AS1 to miR-612. Statistical analysis was carried out by using t test for comparison between two groups, one-way analysis of variance for comparison among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Compared with the miR-NC group, miR-612 group showed significantly decreased cellular proliferative ability, number of migratory cells and invasive cells (all P < 0.05) , but a significantly increased apoptosis rate ( P < 0.05) . The relative expression of miR-612 ( F = 150.78, P < 0.001) , cellular proliferative activity at 24, 48, 72 hours (all P < 0.05) , apoptosis rate and number of migratory and invasive cells (all P < 0.05) significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group. Compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased expression of miR-612 and apoptosis rates, but significantly decreased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly decreased expression of miR-612 and apoptosis rates, but significantly increased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) . Western blot analysis showed that the relative protein expression of cyclinD1, p21, Bcl-2, Bax, MMP-2 and MMP-9 significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group (all P < 0.001) ; compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) . After co-transfection with complementary sequences, the fluorescence activity was significantly lower in the miR-612 overexpression group than in the overexpression control group ( t = 21.19, P < 0.001) ; after co-transfection with non-complementary sequences, no significant difference was observed in the fluorescence activity between the miR-612 overexpression group and overexpression control group ( t = 0.28, P = 0.78) . Conclusion:lncRNA LEF1-AS1 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, likely by targeting miR-612.
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Objective To explore the expression and significance of signal transducer and activator of transcription 3 (Stat 3), glucose transporter protein 1 (GluT-1) and proliferation cell nuclear antigen (PC NA) in lesions of condylomata acuminata (CA). Methods SP immunohistochemistry method was used to measure the expression of Stat 3, GluT-1 and PCNA in tissue samples from 40 cases of CA and 20 normal skin controls. Results The positivity rates of Stat 3, GluT-1 and PCNA were 85.0% (34/40), 87.5% (35/40) and 85.0%(34/40), respectively in CA tissue, 35.0% (7/20), 30.0% (6/20)and 55.0% (11/20),respectively in the control tissue; statistical difference was observed in these rates between the two groups (all P ＜ 0.05). The expression intensity of Stat 3, GluT-1 and PCNA was also higher in CA tissue than that in the controls. In addition, the expression intensity of PCNA was correlated with that of Stat 3 and GluT-1in CA tissue (both P＜ 0.05). Conclusions There is an overexpression of Star 3, GluT-1 and PCNA in CA tissue, and the overexpression of Stat 3 and GluT-1 may be associated with the over-proliferation of CA tissue.