Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice / 中华烧伤杂志

Objective: To investigate the role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice. Methods: The experimental research methods were applied. Eighty-six C57BL/6J male mice (hereinafter briefly referred to as wild-type mice) aged 8-12 weeks were selected for the following experiments. Vγ4 T cells were isolated from axillary lymph nodes of five wild-type mice for the following experiments. Intraperitoneal injection of rapamycin for 42 mice was performed to establish rapamycin-treated mice model for the following experiments. Eighteen wild-type mice were divided into normal control group without any treatment, trauma only group, and trauma+CC chemokine ligand 20 (CCL20) inhibitor group according to the random number table (the same grouping method below), with 6 mice in each group. The full-thickness skin defect wound was made on the back of mice in the latter two groups (the same wound model below), and mice in trauma+CCL20 inhibitor group were continuously injected subcutaneously with CCL20 inhibitor at the wound edge for 3 days after injury. Another 6 rapamycin-treated mice were used to establish wound model as rapamycin+trauma group. On post injury day (PID) 3, the epidermal cells of the skin tissue around the wound of each trauma mice were extracted by enzyme digestion, and the percentage of Vγ4 T cells in the epidermal cells was detected by flow cytometry. In normal control group, the epidermal cells of the normal skin tissue in the back of mice were taken at the appropriate time point for detection as above. Five wild-type mice were used to establish wound models. On PID 3, the epidermal cells were extracted from the skin tissue around the wound. The cell populations were divided into Vγ4 T cells, Vγ3 T cells, and γδ negative cells by fluorescence-activated cell sorter, which were set as Vγ4 T cell group, Vγ3 T cell group, and γδ negative cell group (with cells in each group being mixed with B16 mouse melanoma cells), respectively. B16 mouse melanoma cells were used as melanoma cell control group. The expression of interleukin-22 (IL-22) mRNA in cells of each group was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with the number of samples being 6. Thirty rapamycin-treated mice were used to establish wound models, which were divided into Vγ4 T cell only group and Vγ4 T cell+IL-22 inhibitor group performed with corresponding injections and rapamycin control group injected with phosphate buffer solution (PBS) immediately after injury, with 10 mice in each group. Another 10 wild-type mice were taken to establish wound models and injected with PBS as wild-type control group. Mice in each group were injected continuously for 6 days. The percentage of wound area of mice in the four groups was calculated on PID 1, 2, 3, 4, 5, and 6 after injection on the same day. Six wild-type mice and 6 rapamycin-treated mice were taken respectively to establish wound models as wild-type group and rapamycin group. On PID 3, the mRNA and protein expressions of IL-22 and CCL20 in the peri-wound epidermis tissue of mice in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively. The Vγ4 T cells were divided into normal control group without any treatment and rapamycin-treated rapamycin group. After being cultured for 24 hours, the mRNA and protein expressions of IL-22 of cells in the two groups were detected by real-time fluorescence quantitative RT-PCR and Western blotting, respectively, with the number of samples being 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, Bonferroni method, Kruskal-Wallis H test, and Wilcoxon rank sum test. Results: The percentage of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in trauma only group on PID 3 was 0.66% (0.52%, 0.81%), which was significantly higher than 0.09% (0.04%, 0.14%) in the epidermal cells of the normal skin tissue of mice in normal control group (Z=4.31, P<0.01). The percentages of Vγ4 T cells in the epidermal cells of the skin tissue around the wound of mice in rapamycin+trauma group and trauma+CCL20 inhibitor group on PID 3 were 0.25% (0.16%, 0.37%) and 0.24% (0.17%, 0.35%), respectively, which were significantly lower than that in trauma only group (with Z values of 2.27 and 2.25, respectively, P<0.05). The mRNA expression level of IL-22 of cells in Vγ4 T cell group was significantly higher than that in Vγ3 T cell group, γδ negative cell group, and melanoma cell control group (with Z values of 2.96, 2.45, and 3.41, respectively, P<0.05 or P<0.01). Compared with that in wild-type control group, the percentage of wound area of mice in rapamycin control group increased significantly on PID 1-6 (P<0.01), the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 1 and PID 3-6 (P<0.05 or P<0.01). Compared with that in rapamycin control group, the percentage of wound area of mice in Vγ4 T cell only group decreased significantly on PID 1-6 (P<0.05 or P<0.01). Compared with that in Vγ4 T cell only group, the percentage of wound area of mice in Vγ4 T cell+IL-22 inhibitor group increased significantly on PID 3-6 (P<0.05 or P<0.01). On PID 3, compared with those in wild-type group, the expression levels of IL-22 protein and mRNA (with t values of -7.82 and -5.04, respectively, P<0.01) and CCL20 protein and mRNA (with t values of -7.12 and -5.73, respectively, P<0.01) were decreased significantly in the peri-wound epidermis tissue of mice in rapamycin group. After being cultured for 24 hours, the expression levels of IL-22 protein and mRNA in Vγ4 T cells in rapamycin group were significantly lower than those in normal control group (with t values of -7.75 and -6.04, respectively, P<0.01). Conclusions: In mice with full-thickness skin defects, rapamycin may impair the CCL20 chemotactic system by inhibiting the expression of CCL20, leading to a decrease in the recruitment of Vγ4 T cells to the epidermis, and at the same time inhibit the secretion of IL-22 by Vγ4 T cells, thereby slowing the wound healing rate.

Advance in mechanism study of inflammation in post-ischemic stroke and opportunity of new drug development / 中国药理学通报

Inflammation mediated by the damage-associated molecular patterns (DAMPs) moleculesdominates the pathological process of ischemic stroke in the middle and late stages. In this process, the activation of immune cells and subsequent production of various inflammatory cytokines, chemokines and other cytotoxic mediators are the key factors that trigger inflammation injuries after ischemia. Meanwhile, this mechanism also induces the formation of immune cells with anti-inflammatory activities and tissue repair properties, boosting the regeneration of damaged never system. DAMPs-TLR-NF-κB inflammatory activation chain as the main axis of new anti-stroke drug approaches has become a direction to explore the novel treatments of stroke, which also provides a rational basis for anti-inflammatory therapy in clinical practice for stroke patients. In this paper, we highlighted the current status on the mechanism study with focus on the activation chain of macrophage and microglia involved in DAMPs-TLR-NF-κB in inflammation after stroke and discussed the future development in searching the potential new treatments and therapeutic agents which could be applied in clinics.

Therapeutic effects of total saponins of Panax notoginseng on atherosclerosis in mice / 中国药理学通报

Aim To investigate the therapeutic effects of total saponins of Panax notoginseng( PNS) on ather-osclerosis( AS) in ApoE knockout mice. Methods According to TC level, ApoE knockout mice were ran-domly assigned into six groups. Control group was fed with normal diet, and the other groups were fed with high-fat diet. After 16 weeks, mouse serum and aortas were harvested. The formation of atherosclerotic plaque was analyzed by oil red staining, the proportion of pathological lesion and the apoptosis of endothelial cells in left ventricular outflow tract were analyzed by HE staining and TUNEL. The serum level of lipids profiles, oxLDL-C were detected, and the mRNA ex-pressions of ICAM-1, VCAM-1, iNOS, eNOS, IL-1, IL-6 and TNF-α were detected by qPCR. Results In model group, the serum content of TC, LDL-C and HDL-C significantly increased(P<0.01); the area of atherosclerotic plaque significantly increased ( P <0.01) ; and the apoptosis of endothelial cells and the proportion of pathological lesion of left ventricular out- flow tract significantly increased(P<0.01). Also, the mRNA expression of ICAM-1, VCAM-1, iNOS, IL-1, IL-6 and TNF-α in model group increased. Compared with model group, the serum content of TC, LDL-C and HDL-C decreased after administration of PNS. The serum content of oxLDL-C was significantly reduced in PNS groups( P <0.01, P <0.05 ) . The apoptosis of endothelial cells significantly declined as well ( P <0.01, P <0.05 ) . The area of atherosclerotic plaque decreased after administration of PNS. The mRNA ex-pression of ICAM-1, VCAM-1, iNOS, IL-1, IL-6 and TNF-α in PNS groups were down-regulated. Conclu-sions In ApoE-KO mouse model, PNS plays a role in the therapy of AS, which may be due to its modulating lipid metabolism, protecting vascular endothelium, de-creasing inflammation and inhibiting adhesion of im-mune cells.

Expression characteristics of the USP24 gene in the mouse testis during spermatogenesis / 中华男科学杂志

Objective@#To investigate the expression characteristics of the USP24 gene in the mouse testis and its role in spermatogenesis.@*METHODS@#We examined the expression characteristics of USP24 in the testis tissues of wild-type mice at different postnatal weeks (PNW) and androgen receptor (AR)-knockout (ARKO) adult mice using real-time quantitative PCR and immunofluorescence, and detected the transcriptional activity of the USP24 promoter by dual-luciferase reporter gene assay.@*RESULTS@#The expression of the USP24 gene was low in the testis tissue of the wild-type mice at PNW 1, increased dramatically at PNW 3 and stayed at a similar level till PNW 8. The USP24 protein was located mainly in the cytoplasm of Sertoli and spermatogenic cells. Compared with the wild-type, the adult ARKO mice showed a decreased expression of USP24 localized in the posterior head and mid-piece of the mature sperm in the testis. Dual-luciferase reporter gene assay showed that the transcriptional activity of the USP24 promoter was increased after testosterone stimulation.@*CONCLUSIONS@#The increased expression of the USP24 gene was associated with the initiation of sexual development, and the USP24 protein was expressed in the mature sperm of the mice. USP24 is an AR-target gene, which may be involved in the regulation of spermatogenesis in mice.

Clinical study of perineural invasion in patients with rectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of perineural invasion(PNI) in rectal cancer.</p><p><b>METHODS</b>Clinical data of 204 patients undergoing resection of low rectal cancer from January 2003 to January 2005 at the First People's Hospital of Chenzhou were analyzed retrospectively. Paraffin sections of surgical specimens from all the patients who underwent resection of low rectal cancer were stained with HE. PNI-positive was defined as infiltration of carcinoma cell into the perineurium or neural fascia. The association of PNI with clinicopathologic features and prognosis of rectal cancer was analyzed.</p><p><b>RESULTS</b>PNI was positive in 31.9%(65/204) of the patients. The tumor size, depth of invasion, lymph node metastasis, TNM stage, tumor growth pattern, histologic grade, tumor resection were significantly associated with PNI. The overall survival time of the PNI-positive patients was shorter than that of the PNI-negative patients[(43.8±1.5) months vs.(57.2±1.5) months, P<0.01]. Furthermore, the overall survival time of the PNI-positive stage II( patients was shorter than that of the stage III( patients [(46.5±3.2) months vs. (55.7±1.2) months, P<0.05].</p><p><b>CONCLUSION</b>PNI can be used as one of the indicators to predict the prognosis of patients with rectal cancer.</p>

Serum proteomic analysis on metastasis-associated proteins of hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the serum proteins associated with the metastasis of hepatocellular carcinoma (HCC) using a comparative proteomic approach.</p><p><b>METHODS</b>The serum samples of HCC patients with the same disease background were divided into metastatic (n=20) and non-metastatic (n=20) groups. The proteins extracted from the patients and 20 normal subjects, after depletion of the highly abundant proteins, underwent two-dimensional gel electrophoresis (2-DE). Comparative analyses of the 2-DE protein patterns between the 3 groups were conducted using a computerized image analysis system. The proteins with statistically significant differential expression between the metastatic and non-metastatic patients were identified by mass spectrometry. Western blotting was performed to examine the differential expression of the candidate proteins.</p><p><b>RESULTS</b>Four protein spots were identified by mass spectrometry among the 12 differentially expressed protein spots in the serum samples of HCC patients with intrahepatic metastasis, and confirmed by searching in MASCOT database. Of the 4 proteins, cytokeratin 9 (CK9) was up-regulated by 2 folds, and inter-alpha (globulin) inhibitor H4, complement factor H-related protein 1 precursor (FHR-1), and apolipoprotein E were down-regulated by 2 folds. CK9 was found to be specifically over-expressed in the metastatic group in comparison with the non-metastatic group, as confirmed by Western blotting.</p><p><b>CONCLUSION</b>The metastasis of HCC might be correlated to the specific variation of protein expression profiles. The overexpression of CK9 may play a crucial role in HCC metastasis, and can be used as a potential serum marker for predicting HCC metastasis.</p>