Mechanism of Guishenwan on Thin Endometrium Rats Based on Network Pharmacology / 中国实验方剂学杂志

Objective:This study explores the key core targets of Guishenwan in the treatment of thin endometrium and related signaling pathways through the method of network pharmacology，and further uses animal experiments to verify the obtained targets and verify that Guishenwan are effective for thin endometrium. Method:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） was used to retrieve the effective chemical components，active component targets and target abbreviations of the eight Chinese medicines in Guishenwan，the GeneCards database and Online Mendelian Inheritance in Man（OMIM）database were used to retrieve thin endometrial related targets gene.Use Wayne software to take the intersection of the drug target of Guishenwan and the disease target of the thin endometrium，and import the intersection target into the STRING database and Cytoscape 3.7.2 software for visual analysis to obtain the "drug-disease" protein protein interaction（PPI） network， then input the intersection target into Enrichr database and DAVID database for gene ontology（GO） enrichment analysis and Kyoto encyclopedia of genes and genomes（KEGG） enrichment analysis. Using the obtained possible core and key targets as the theoretical basis，a thin endometrial model in rats was established. After Guishenwan and estrogen intervention for 21 days，the endometrial thickness of rats was observed by hematoxylin-eosin staining（HE） staining. Western blot and quantitative real time polymerase chain reaction（Real-time PCR） detect the protein and mRNA expression levels of the four core key targets of epidermal growth factor receptor（EGFR），matrix metalloproteinase 9（MMP9），interleukin-1beita（IL-1<italic>β</italic>） and mitogen activated protein kinase 14（MAPK14）. Result:The Venn software obtained 130 intersection targets in total， imported 130 intersection targets into the STRING database and Cytoscape database，and obtained a protein interaction network diagram including 33 nodes and 107 edges. DAVID 6.8 database for GO analysis. The function annotation analysis involving 167 biological processes（BP），22 cell components（CC），39 molecular functions（MF）. DAVID 6.8 database for KEGG enrichment analysis， and thin endometrium related A total of 34 pathways. Western blot and Real-time PCR were used to analyze the expression results of EGFR，MMP9，IL-1<italic>β</italic>，MAPK14 protein and genes in the endometrial tissues of the 6 groups of rats. Guishenwan can enhance the expression of EGFR，MMP9，IL-1<italic>β</italic>，MAPK14 protein and mRNA on the thin endometrium. Conclusion:According to the theoretical analysis of network pharmacology and the results of animal experiments，it is found that Guishenwan can effectively improve the related indicators of thin endometrium，and promote the expression of EGFR，MMP9，IL-1<italic>β</italic>，MAPK14 protein and genes. The intimal tissue proliferates and improves the symptoms of thin intima.

Expression of abnormal spindle-likemicrocephaly-associated in hepatocellular carcinoma and its prognostic relationship after liver transplantation for hepatocellular carcinoma / 中华器官移植杂志

Objective:To explore the expression of abnormal spindle-like microcephaly-associated (ASPM) in liver cancer tissues and clarify its prognostic relationship with clinicopathological features of liver cancer after liver transplantation.Methods:Immunohistochemistry was employed for detecting the expression of ASPM in 72 liver cancer tissues and 36 adjacent tissues of liver cancer liver transplant recipients fulfilling the Hangzhou criterion. In conjunctions with clinicopathological data, the correlation between the expression level of ASPM in liver cancer tissues and the clinicopathological characteristics and the post-transplantation prognosis for liver cancer were statistically analyzed.Results:During a median follow-up period of 29 months, 20 patients relapsed and 8 died after transplantation. Immunohistochemical results indicated that the high-expression rates of ASPM were 58.3% and 25.0% in liver cancer and adjacent tissues ( P=0.001). The difference was statistically significant. The high-expression rate of ASPM was significantly higher in liver cancer tissues than that in adjacent tissues. The expression level of ASPM was not correlated with gender, age, smoking/alcoholic history, hepatitis history, preoperative level of alpha-fetoprotein (AFP), tumor size, tumor load or vascular tumor thrombus ( P>0.05). And the postoperative high-expression rates of ASPM were 51.0% and 76.2% in pathological differentiation type Ⅰ-Ⅱ and Ⅲ-Ⅳ groups ( P=0.049). The difference was statistically significant. The wrose pathological differentiation type of liver cancer, the higher expression level of ASPM in liver cancer tissue. In liver cancer tissues, the overall 1/3/5-year survival rates of ASPM high/low-expression group were 97.6%, 80.6%, 80.6% and 93.3%, 89.7% and 89.7% respectively ( P>0.05). There was no statistical significance. And 1/3/5-year long-term disease-free survival rates were 78.6%, 55.5%, 55.5% and 86.3%, 86.3% and 86.3% respectively ( P=0.036). The difference was statistically significant. The disease-free survival rate was lower in ASPM high-expression group and post-transplantation prognosis was worse. Conclusions:The expression of ASPM is significantly higher in liver cancer tissues than that in adjacent tissues. And the expression level of ASPM in liver cancer tissues is correlated with pathological differentiation types of liver cancer and has an impact on tumor-free survival of patients after liver transplantation for liver cancer.

High incidence of asymptomatic SARS-CoV-2 infection, Chongqing, China

BackgroundSARS-CoV-2 has been a global pandemic, but the emergence of asymptomatic patients has caused difficulties in the prevention of the epidemic. Therefore, it is significant to understand the epidemiological characteristics of asymptomatic patients with SARS-CoV-2 infection. MethodsIn this single-center, retrospective and observational study, we collected data from 167 patients with SARS-CoV-2 infection treated in Chongqing Public Health Medical Center (Chongqing, China) from January to March 2020. The epidemiological characteristics and variable of these patients were collected and analyzed. Findings82.04% of the SARS-CoV-2 infected patients had a travel history in Wuhan or a history of contact with returnees from Wuhan, showing typical characteristics of imported cases, and the proportion of severe Covid-19 patients was 13.2%, of which 59% were imported from Wuhan. For the patients who was returnees from Wuhan, 18.1% was asymptomatic patients. In different infection periods, compared with the proportion after 1/31/2020, the proportion of asymptomatic patient among SARS-CoV-2 infected patient was higher(19% VS 1.5%). In different age groups, the proportion of asymptomatic patient was the highest(28.6%) in children group under 14, next in elder group over 70 (27.3%). Compared with mild and common Covid-19 patients, the mean latency of asymptomatic was longer (11.25 days VS 8.86 days), but the hospital length of stay was shorter (14.3 days VS 16.96 days). ConclusionThe SARS-CoV-2 prevention needs to focus on the screening of asymptomatic patients in the community with a history of contact with the imported population, especially for children and the elderly population.

In-situ split liver transplantation for pediatric recipient: a single-center experience / 中华器官移植杂志

Objective To evaluate the efficacy of in-situ split liver transplantation (ISSLT) in children.Methods From June 2015 to August 2018,10 liver grafts from DBD were split in-situ.All the donors were male,and the median age of the donors was 28.5 year old (18-48 year).One left half graft and 9 left lateral lobe grafts (including 2 reduced size grafts) were transplanted to 10 pediatric recipients.Four grafts were transplanted in our center,and the rest 6 grafts were shared to other two transplant center.The primary diseases of the recipients included biliary atresia (8/10),hepatic sinus obstruction syndrome (1/10) and Alagille syndrome (1/10).The median age of the recipients was 10 month (7 month-11 year),and the mean body weight was 9.8 ± 6.6 kg (5-28 kg).Results All liver grafts were split in-situ.The mean split time of liver grafts was 88.5 ± 18.9 min.The mean weight of split grafts was 336.7-± 85.4 g.All recipients were subjected to piggyback liver transplantation.Operation time was 542.5 ± 112.1 min.Anhepatic time was 52.0 ±-13.5 min.GRWR was (3.98 ±0.96)％.GRWR of two cases was more than 5％,so segment Ⅲ was partially reduced.During the follow-up period,9 cases were alive and 1 case died due to multiple organ failure 1 day after liver transplantation.Conclusions ISSLT can enlarge the graft pool for children and achieve good results.

Role of estrogen, estrogen receptors, and aromatase in the pathogenesis of uterine adenomyosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the role of estrogen (E2), estrogen receptor (ER) and aromatase (P450arom) in the pathogenesis of uterine adenomyosis.</p><p><b>METHODS</b>Paraffin-embedded specimens of the uterine tissue from patients with uterine adenomyosis and patients with cervical lesions (CIN; control) were examined for expressions of E2, ER and P450arom by immunohistochemistry and ELISA. The cells isolated from the lesions of patients with adenomyosis were cultured in vitro, and the changes in cell growth in response to treatments with E2, ER inhibitor, ER inhibitor + E2, estrogen deprivation, and estrogen deprivation+ ICI182780 were assessed using CCK-8 method.</p><p><b>RESULTS</b>The expression levels of E2, ER, and P450arom were significantly higher in adenomyosis ectopic lesions and eutopic endometrium than in the myometrium and endometrium in the control group (P<0.05); no significant difference in E2 and P450arom expressions was found between adenomyosis ectopic lesions and eutopic endometrium (P>0.05), while the expression levels of ER in ectopic lesions was significantly higher than that in eutopic endometrium. The cell inhibition rates were similar between ER inhibitor group and ER inhibitor + Estrogen activation group (P>0.05), and was significantly higher in estrogen deprivation+ ER inhibitor group than in estrogen deprivation group (P<0.05).</p><p><b>CONCLUSION</b>The high expression levels of E2, ER, and P450arom in adenomyosis ectopic lesions and eutopic endometrium promote uterine adenomyosis cell proliferation, in which process E2 combines with ER to execute its biological effect; ER also promotes the occurrence and development of uterine adenomyosis through other pathways.</p>

ABCG2 protects kidney side population cells from hypoxia/reoxygenation injury through activation of the MEK/ERK pathway.

Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.

Impact of HLA-G 14-bp polymorphism on acute rejection and cytomegalovirus infection in kidney transplant recipients from northwestern China.

Human leukocyte antigen (HLA)-G plays an important role in promoting transplant tolerance and helping human cytomegalovirus (CMV) to subvert host defenses. Strong evidence suggests that HLA-G 14-bp insertion/deletion polymorphism influences the stability of HLA-G mRNAs and levels of protein expression. We hypothesized that HLA-G 14-bp polymorphism of recipients has an influence on the risk of acute rejection (AR) and CMV infection. We investigated the impact of HLA-G 14-bp polymorphism on a total of 363 unrelated Chinese Han individuals who included 42 kidney transplant recipients with AR, 43 recipients with CMV infection, 102 recipients with stable allograft function (STA), and 176 healthy controls (HC). No statistically significant difference was found between all kidney transplant patients and HC (P=0.149). But, our data showed an increased frequency of homozygous genotype +14/+14 bp (P(c)=0.004) and allele +14 bp (P(c)=0.002) in patients with AR when compared with STA, with the odds ratio of 3.17 and 2.28, respectively. Moreover, we found that the frequency of the -14/-14 bp genotype (P(c)=0.008) and the -14 bp allele (P(c)=0.016) was increased in patients with CMV infection when compared with STA, with the OR of 2.66 and 1.96, respectively. Multivariate analysis further demonstrated that HLA-G homozygous +14 bp and -14 bp genotypes were an independent risk factor for allograft rejection and CMV infection, respectively. In conclusion, this study identified an important genetic risk factor for acute allograft rejection, and it was the first to show a significant correlation between HLA-G 14-bp polymorphism and CMV infection after kidney transplantation from northwestern China.

[The influence of rapamycin on BTLA expression of human peripheral blood T lymphocytes].

AIM: To explore the effect of rapamycin (RPM) on the expression of B and T lymphocyte attenuate (BTLA) on human peripheral blood T lymphocytes, providing a experimental basis for application of RPM to organ transplantation and autoimmune diseases. METHODS: Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and then the T cells were isolated from PBMCs with immunomagnetic beads. Concanavalin A (Con A) used to stimulate and activate the human peripheral blood T cells. The proliferation of T cells was detected by MTT colorimetry. The levels of IL-2 and IFN-γ in cell culture supernatant were detected by ELISA. The expression of BTLA on T cells was assayed by Flow cytometry. RESULTS: The expression of BTLA on T cells treated with various concentrations (10-1 000 ng/L) of RPM had no significant difference, while had significant difference (P<0.01) by compared with RPM non-treatment group. ELISA detection manifested that compared with the untreated group different concentrations of RPM could significantly inhibit the IL-2 and IFN-γ secretion and significantly inhibited T lymphocyte proliferation (P<0.01). CONCLUSION: RPM has little effect on expression BTLA, but has stronger inhibition on lymphocyte proliferation and inflammatory cytokines secretion. Suggesting that RPM is suitable for the treated of organ transplant rejection and autoimmune diseases.

[Evaluation of immune status of kidney transplant recipients by combined HLA-G5 and sCD30].

OBJECTIVE: to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. METHODS: from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. RESULTS: the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L. CONCLUSION: the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells.

OBJECTIVE: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4(+) CD25(+) regulatory T (Treg) cells, in order to inform further studies in Treg cell function. METHODS: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4(+) CD25(+) Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4(+) CD25(+) Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4(+) CD25(+) Treg cells in the in vitro proliferation experiments. RESULTS: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4(+) CD25(+) Treg cells, however, obtained with both methods have similar immunosuppressive capacities. CONCLUSION: The result suggests that both methods can be used in isolating CD4(+) CD25(+) Treg cells, and one can select the best method according to specific needs and availability of the methodologies.