Effects of DNMT3A on mouse cardiac fibroblast proliferation and migration under high glucose environment北大核心CSCD

Aim To investigate the effect of DNA methyltransferase 3A (DNMT3A) on the proliferation and migration of cardiac fibroblasts (CFs) in C57 mice under high glucose environment. Methods The hearts of C57 mice were taken from 1 to 3 days. After cutting and digesting, CFs were extracted by differential adherance centrifugattion and observed under microscope. After cell attachment, the cells were cultured under low glucose (5.5 mmol • L -1) medium and high glucose (33.0 mmol • L-1) medium and treated using DNMT3A lentivirus ( to silence the DNMT3A gene) . qRT-PCR was used to detect mRNA expression of DNMT3A, a-smooth muscle actin ( α-SMA ) and type I collagen procollagen Al ( Coll Al ) , the protein expression of DNMT3A, α-SMA and CollAl was assayed by Western blot, cell proliferation activity was detected by CCK-8 assay and MTT assay,cell migration ability was detected by scratch assay and Transwell experiment. Results Compared with the low glucose group,the high glucose environment resulted in up-regulated DNMT3A expression in CFs, increased collagen fiber deposition, and promoted CFS proliferation and migration. The expression of DNMT3A was down-regulated in CFs treated with DNMT3A lentivirus under high glucose condition, α-SMA, CollAl expression was down regulated, and cell proliferation and migration ability decreased. Conclusions Silencing of DN-MT3A could inhibit the proliferation and migration of CFs under high glucose environment, suggesting that DNMT3A may be a key regulatory point in the develop¬ment of diabetic cardiomyopathy. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Isoindolone derivative QSN-IOc induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition

Aim： 2-（4,6-Dimethoxy-l,3-dioxoisoindolin-2-yl） ethyl 2-chloroacetate （QSN-IOc） is one of isoindolone derivatives with antiproliferative activity against human umbilical vein endothelial cells （HUVECs）. The aim of this study was to investigate its antitumor activity in vitro and anti-angiogenic effects in vitro and in vivo. Methods： K562 leukemic cells and HUVECs were used for in vitro studies. Cell viability was examined using MTT assay. Cell apoptosis and mitochondrial transmembrane potential （Δψm） were detected with flow cytometry. Tube formation and migration of HUVECs were studied using two-dimensional Matrigel assay and wound-healing migration assay, respectively. VEGF levels were analyzed with RT-PCR and Western blotting. A zebrafish embryo model was used for in vivo anti-angiogenic studies. The molecular mechanisms for apoptosis in K562 cells and antiangiogenesis were measured with Western blotting. Results： In antitumor activity studies, QSN-IOc suppressed the viability of K562 cells and induced apoptosis in dose- and time- dependent manners. Furthermore, QSN-IOc dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway. In anti-angiogenic activity studies, QSN-IOc suppressed the viability of HUVECs and induced apoptosis in dose dependent manners. QSN-IOc treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo. Furthermore, QSN-IOc dose-dependently suppressed the phosphorylation of AKT and GSK313 in both HUVECs and K562 cells. Conclusion： QSN-IOc is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/ AKT/GSK313 signaling pathway.     

Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402北大核心CSCD

Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 0～8 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Role and mechanism of NR1D1 in proliferation and migration of vascular adventitial fibroblasts北大核心CSCD

Aim To explore the role and mechanism of nuclear receptor subfamily 1,group D,member 1(NR1D1)in the proliferation and migration of mouse adventitial fibroblasts(AFs). Methods Primary AFs isolated from C57BL/6J mice were cultured. Adenovirus carrying Nr1d1 gene was used to overexpress NR1D1 in AFs. The expression of β-catenin was restored by SKL2001. Proliferating cell nuclear antigen(Ki-67)immunofluorescence staining and CCK-8 staining were used to determine cell proliferation,and scratch test was used to determine cell migration. qPCR was used to determine the mRNA level of Nr1d1. Western blot was used to determine the protein levels of NR1D1 and β-catenin. To investigate the role of NR1D1 in intimal hyperplasia,20 male wild type C57BL/6J mice were randomly divided into sham group,carotid artery endothelial injury,sham+SR9009(NR1D1 agonist)group and carotid artery endothelial injury+SR9009(n=5 in each group). They were treated with DMSO or SR9009(100 mg·kg-1·d-1)via intraperitoneal injection for 14 days after operation,respectively. The degree of carotid intimal hyperplasia was measured by HE staining 28 days after operation. Results NR1D1 overexpression significantly reduced the percentage of Ki-67-positive cells(P<0.01),total cell number(P<0.01)and slowed down the rate of wound-healing(P<0.01). NR1D1 overexpression significantly inhibited the expression of β-catenin(P<0.05). After the expression of β-catenin was restored by SKL2001,the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs were abolished(P<0.01). Enhanced activity of NR1D1 significantly ameliorated intimal hyperplasia after carotid endothelial injury(P<0.01). Conclusion NR1D1 may inhibit the proliferation and migration of AFs via suppressing the expression of β-catenin. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Microvesicles secreted from human multiple myeloma cells promote angiogenesis

Aim： To investigate whether human multiple myeloma （MM） cells secrete microvesicles （MVs） and whether the MVs secreted from MM cells （MM-MVs） promote angiogenesis. Methods： RPMI8226 human MM cells and EA.hy926 human umbilical vein cells were used. MVs isolated from RPMI 8226 cells were characterized under laser confocal microscopy, electron microscopy and with flow cytometry. The fusion of MM-MVs and EA.hy926 cells was studied under confocal microscopy, and the transfer of CD138 to EA.hy926 cells was demonstrated with flow cytometry. The proliferation, invasion and tube formation of EA.hy926 cells in vitro were evaluated using M]-r, transwell migration and tube formation assays, respectively. The vasculization of EA.hy926 cells in vivo was studied using Matrigel plug assay. The expression of IL-6 and VEGF was analyzed with PCR and ELISA. Results： MM-MVs from the RPMI 8226 cells had the characteristic cup-shape with diameter of 100-1000 nm. Most of the MM-MVs expressed phosphatidylserine and the myeloma cell marker CD138, confirming that they were derived from myeloma cells. After added to EA.hy926 cells, the MM-MVs transferred CD138 to the endothelial cells and significantly stimulated the endothelial cells to proliferate, invade, secrete IL-6 and VEGF, two key angiogenic factors of myeloma, and form tubes in vitro and in vivo. Conclusion： Our results confirm the presence of MVs in MM cells and support the idea that MM-MVs are newfound mediators for myeloma angiogenesis and may serve as a therapeutic target to treat MM.     

Targeting fibroblast activation protein inhibits endothelial-mesenchymal transition by affecting cancer-associated fibroblasts derived exosomes北大核心CSCD

Aim To investigate whether targeted inhibition of fibroblast activation protein (FAP) can inhibit the endothelial-to-mesenchymal transition (EndMT) of vascular endothelial cells by affecting exosomes (Exo) of cancer-associated fibroblasts (CAFs) and explore the underlying mechanisms. Methods Primary CAFs and peri-tumor fibroblasts (PTFs) were obtained from lung cancer and peri-cancer tissues, and CAFs-exo and PTFs-exo were collected from culture medium, respectively. Exosomes from CAFs treated with specific FAP inhibitor (3.3 nmol • L-1 SP13786) for 24 h were named as Anti-FAP-exo. HMEC-1 cells were incubated in equal volumes of RPMI 1640, PTFs-exo, CAFs-exo and anti-FAP-exo respectively and named as control group, PTF group, C AF group and anti-FAP group. The scratch assay, Transwell invasion assay and angiogenesis assay were used to detect the migration ability, invasion ability and angiogenesis ability of HMEC-1 cells. Immunofluorescence, immunohistochemistry and Western blot were used to detect EndMT-associated protein expression. Results The migration ability, invasion ability and angiogenesis ability of HMEC-1 cells of CAF group were significantly higher than those of PTF group, whereas there was no significant difference between that of anti-FAP group and PTF group. HMEC-1 cells of CAF group had higher expression of α-SMA, SM22α, p-Stat3 and Snail, and lower expression of CD31 and VE-cadherin than that of PTF group. In addition, HMEC-1 cells of Anti-FAP group had lower expression of α-SMA, SM22α, pStat3 and Snail, and higher expression of CD31 and VE-cadherin than that of CAF group. Conclusions Specific inhibition of FAP could indirectly inhibit the migration ability, invasion ability and angiogenesis ability of vascular endothelial cells via affecting CAFs-exo and Stat3-snail-EndMT pathway may be the potential mechanism. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Role and possible mechanism of estrogen receptor α down-regulation leading to damage of TM4 Sertoli cell connectivity in mice北大核心CSCD

Aim To investigate the role of autophagy in the dysfunction of testicular TM4 cell junction induced by ERα down-regulation. Methods TM4 cells were treated with different concentrations of E R a inhibitor ICI182780 (ICI), and the proliferative activity of TM4 cells was detected by CCK-8 method. The number and morphological changes of TM4 cells were observed by light microscope. The levels of E R a, junction function related proteins and autophagy marker proteins were detected by Western blot. The expression and localization of Cx43 were detected by immunofluorescence staining. The cells were treated with chloroquine (CQ) and ICI for 24 h. The expression levels of autophagy and junction function related proteins were detected by Western blot. Results When ICI concentration was 50 nmol • L ~ or above, the cell viability decreased significantly. The increase of cell vacuoles in ICI group was observed by light microscope. Compared with normal control group, the protein expression levels of E R a, ZO-1, occludin, claudin-11, p-catenin and Cx43 in ICI groups significantly dropped, while the expression levels of N-cadherin and E-cadherin had no significant changes; LC3 II significantly rose, while p62 expression significantly fell. The results of immunofluorescence showed that the fluorescence expression of Cx43 in ICI group decreased significantly, but the position of CX43 did not change significantly. Compared with ICI group, the expression levels of LC3 II, p62, Cx43, ZO-1 and β-Catenin significantly increased. Conclusions The down-regulation of E R a leads to damage of TM4 cell junction function, which may be related to the activation of autophagy. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

Aim： To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation. Methods： ECV304 cells derived from adult human umbilical vein endothelial cells （HUVEC） were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF （20 or 40 ng/mL） 3 h prior to irradiation. At 48 h post- irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay, Results： c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytom- etry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells. Conclusion： HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.     

Effects of RNA demethylase FTO inhibitor on function ofglioblastoma stem cells北大核心CSCD

Aim To investigate the effect of m6A demethylase FTO inhibitor(FB23-2)on human glioblastoma stem cell activity. Methods The effects of FB23-2 and Temozolomide on GSC were detected by CCK-8 assay and neurosphere formation assay. The effect of FB23-2 on self-renewal of GSC was detected by limited dilution assay in vitro. The effect of FB23-2 on the proliferation of GSC was detected by EdU method. The effect of FB23-2 on apoptosis of glioblastoma stem cells was detected by flow cytometry. Results CCK-8 assay showed that FB23-2 could effectively inhibit the cell viability of GSC with IC50 values of 7.11 μmol·L-1 and 4.63 μmol·L-1,respectively. The size and number of GSC neural sphere in FB23-2 treatment group were significantly reduced compared with control group. In vitro limited dilution experiment showed that FB23-2 effectively inhibited the self-renewal ability of GSC. EdU incorporation experiment showed that compared with the control group,the treatment group decreased to(70.59±13.74)% and(50.33±4.53)%,respectively. The apoptotic rates of the treated group were(12.16±1.90)% and(16.77±1.17)% by flow cytometry. Conclusions FTO inhibitor FB23-2 can effectively inhibit GSC growth,self-renewal and the formation of neural sphere. In addition,FB23-2 can inhibit the proliferation of GSC and induce its apoptosis. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Artesunate inhibits development of esophageal squamous cell carcinoma via regulating expression of Skp2 and P21北大核心CSCD

Aim To clarify the regulatory effect of Artesunate(ART) on tumor cell function and cell cycle in the pathological process of esophageal squamous cell carcinoma(ESCC). Methods KYSE450 and TE14 cells were treated with different concentrations of ART. The cells treated with 0 mg •L-1 ART were used as the control group, and the cells treated with 30 mg•L -1ART were used as the experimental group. MTT assay and cloning formation experiment were used to sense cell growth. Transwell assay was applied to test the invasion and migration capacity of ART treated group and blank group separately,flow cytometry was employed to examine c ell cycle. The Mechanism of Artesunate on Escc by RNA-Seq was detected. Differential Genes We value USIDANG RT-QPCR. Results Compared with the Control Group, The P Roliferation, Invasion and Migration of Escc Cells Were SUPPRSSED CONSIDERABLY AFTER Art Treatment (P <0.01). According to the analysis of sequencing results, ART might inhibit the occurrence and development of tumor by affecting the cell cycle. After ART treatment, the ratio of KYSE450 cells at G1 phase boosted 18.02%. The proportion of KYSE450 cells in G1 phase increased significantly in the back of adding ART(24.12%±2.62% vs 42.14%±0.65%, P<0.01), and the percentage of TE14 cells at G 1phase also increased significantly after ART therapy(36.25%±0.21% vs 42.19%±0.38%, P<0.01). Skp2, a gene closely related to cell cycle regulation, decreased and its downstream target gene P21 increased. Conclusions ART, a Chinese patent medicine, can inhibit the proliferation, invasion and migration of ESCC cells, accelerate apoptosis and cause cell arrest through regulating the expression of Skp2 and P21. This study takes the re-application of Chinese patent medicine which is used against malaria originall y as the innovation point to provide a reference basis for the development of new safety, effective and economical targeted drugs for ESCC. It also provides scientific ideas for the development of a new treatment scheme of radiotherapy combined with ESCC. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

S-allylcysteine,a garlic derivative,suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro

Aim： To investigate the effects of S-allylcysteine （SAC）, a water-soluble garlic derivative, on human ovarian cancer cells in vitro. Methods： Human epithelial ovarian cancer cell line A2780 was tested. Cell proliferation was examined with CCK-8 and colony formation assays. Cell cycle was analyzed with flow cytometry. Cell apoptosis was studied using Hoeohst 33258 staining and Annexin V/PI staining with flow cytometry. The migration and invasion of A2780 cells were examined with transwell and wound healing assays. The expression of relevant proteins was detected with Western blot assays. Results： SAC （1-100 mmol/L） inhibited the proliferation of A2780 cells in dose- and time-dependent manners （the ICsovalue was approximately 25 mmoVL at 48 h, and less than 6.25 mmol/L at 96 h）. Furthermore, SAC dose-dependently inhibited the colony formation of A2780 cells. Treatment of A2780 cells with SAC resulted in G/S phase arrest and induced apoptosis, accompanied by decreased expression of pro-caspase-3, Parp-1 and Bcl-2, and increased expression of active caspase-3 and Bax. SAC treatment significantly reduced the migration of A2780 cells, and markedly decreased the protein expression of Wnt5a, p-AKT and c-Jun, which were the key proteins involved in proliferation and metastasis. Conclusion： SAC suppresses proliferation and induces apoptosis in A2780 ovarian cancer cells in vitro.     

TSPO ligands prevent the proliferation of vascular smooth muscle cells and attenuate neointima formation through AMPK activation

Abnormal growth of the intimal layer of blood vessels(neointima formation)contributes to the progression of atherosclerosis and in-stent restenosis.Recent evidence shows that the 18-kDa translocator protein(TSPO),a mitochondrial membrane protein,is involved in diverse cardiovascular diseases.In this study we investigated the role of endogenous TSPO in neointima formation after angioplasty in vitro and in vivo.We established a vascular injury model in vitro by using platelet-derived growth factor-BB(PDGF-BB)to stimulate rat thoracic aortic smooth muscle cells(A10 cells).We found that treatment with PDGF-BB(1-20 ng/mL)dose-dependently increased TSPO expression in A10 cells,which was blocked in the presence of PKC inhibitor or MAPK inhibitor.Overexpression of TSPO significantly promoted the proliferation and migration in A10 cells,whereas downregulation of TSPO expression by siRNA or treatment with TSPO ligands PK11195 or Ro5-4864(104 nM)produced the opposite effects.Furthermore,we found that PK11195(10−104 nM)dose-dependently activated AMPK in A10 cells.PK11195-induced inhibition on the proliferation and migration of PDGF-BB-treated A10 cells were abolished by compound C(an AMPK-specific inhibitor,103 nM).In rats with balloon-injured carotid arteries,TSPO expression was markedly upregulated in the carotid arteries.Administration of PK11195(3 mg/kg every 3 days,ip),starting from the initial balloon injury and lasting for 2 weeks,greatly attenuated carotid neointima formation by suppressing balloon injury-induced phenotype switching of VSMCs(increasedα-SMA expression).These results suggest that TSPO is a vascular injury-response molecule that promotes VSMC proliferation and migration and is responsible for the neointima formation after vascular injury,which provides a novel therapeutic target for various cardiovascular diseases including atherosclerosis and restenosis.     

MicroRNA-302a promotes neointimal formation following carotid artery injury in mice by targeting PHLPP2 thus increasing Akt signaling

The excessive proliferation and migration of smooth muscle cells(SMCs)play an important role in restenosis following percutaneous coronary interventions.MicroRNAs are able to target various genes and involved in the regulation of diverse cellular processes including cell growth and proliferation.In this study we investigated whether and how MicroRNAs regulated vascular SMC proliferation and vascular remodeling following carotid artery injury in mice.We showed that carotid artery injury-induced neointimal formation was remarkably ameliorated in microRNA(miR)-302 heterozygous mice and SMC-specific miR-302 knockout mice.In contrast,delivery of miR-302a adenovirus to the injured carotid artery enhanced neointimal formation.Upregulation of miR-302a enhanced the proliferation and migration of mouse aorta SMC(MASMC)in vitro by promoting cell cycle transition,whereas miR-302a inhibition caused the opposite results.Moreover,miR-302a promoted Akt activation by corporately decreasing Akt expression and increasing Akt phosphorylation in MASMCs.Application of the Akt inhibitor GSK690693(5μmol/L)counteracted the functions of miR-302a in promoting MASMC proliferation and migration.We further revealed that miR-302a directly targeted at the 3′untranslated region of PH domain and leucine rich repeat protein phosphatase 2(PHLPP2)and negatively regulated PHLPP2 expression.Restoration of PHLPP2 abrogated the effects of miR-302a on Akt activation and MASMC motility.Furthermore,knockdown of PHLPP2 largely abolished the inhibition of neointimal formation that was observed in miR-302 heterozygous mice.Our data demonstrate that miR-302a exacerbates SMC proliferation and restenosis through increasing Akt signaling by targeting PHLPP2.     

Ginsenoside Rgl promotes endothelial progenitor cell migration and proliferation

Aim： To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells （EPCs）.
Methods： EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl （0.1, 0.5, 1.0, and 5.0 μmol/L） and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3.（4,5.dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.
Results： Ginsenoside Rgl promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and timedependent manner. Cell cycle analysis showed that 5.0 μmol/L ofginsenoside Rgl significantly increased the EPC proliferative phase （S phase） and decreased the resting phase （G0/G1 phase）. Ginsenoside Rgl increased vascular endothelial growth factor production. Conclusion： The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Effect of Clkl gene on level of Aβ-induced autophagy in Alzheimer's disease北大核心CSCD

Aim To investigate the effect of siRNA transfection of silencing Clkl gene on autophagy levels in AD model cells. Methods The Clkl gene was silted using siRNA transfection techniques. MTT was used to observe the effects of Aβ2 5 35 and Clkl genes on cell survival. Cellular immunofluorescence was applied to observe the expression of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). Western blot was employed to detect the expression of Clkl, HIF-1α, PI3K/AKT/mTOR, autophagy-related proteins Beclinl,LC3-II/I,and p62. Quantitative realtime polymerase chain reaction (RT-qPCR) was utilized to detect the expression of Beclinl, LC3, p62 mRNA. Results Compared with the NC + NS group, the viability of HT22 cells in the NC + Aβ group was significantly reduced (P < 0.01), while the viability of cells in the siClkl + Aβ group was significantly higher than that in the N C + A β group (P < 0.01). The results of cellular immunofluorescence showed that the expression of LC3 in the NC + Aβ group was significantly higher than that in the NC + NS group (P < 0.01), and the expression of LC3 in the siClkl + Aβ group was significantly lower than that in the NC + Aβ group (P < 0.01). Compared with the NC + NS group, the expression of Beclinl and LC3 -II/I proteins in the NC + Aβ group was significantly raised (P < 0.01), and the expression of p62, PI3 K, p-AKT/AKT, and p-mTOR/mTOR was significantly reduced (P < 0.01). Compared with the NC + Aβ group, the expression of Beclinl and LC3-II/I proteins in the siClkl + Aβ group decreased significantly (P <0.01), and the expression of p62, PI3 K, p-AKT/AKT, and p-mTOR/mTOR proteins increased significantly (P < 0.01). The addition of LY294002 and YC-1 reversed the effect of silencing Clkl on Beclinl, LC3-II/I, p62, PI3K, p-AKT/AKT, p-mTOR/mTOR proteins. RT-qPCR results showed that there was no significant difference in Beclinl, LC3 and p62 mRNA levels in the siClkl + Aβ + YC-1 group compared with the siClkl + Aβ group (P > 0.05), and Beclinl, LC3 and p62 mRNA were consistent with the protein levels in the other groups. Conclusions Silencing Clkl expression can alleviate the decrease in cell viability induced by excessive autophagy of HT22 cells induced by Aβ2 535, and the mechanism may be related to the PI3 K/AKT/mTOR signaling pathway and HIF-1α. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Mechanism of Buyang Huanwu Decoction alleviating inflammatory response after cerebral ischemia/reperfusion injury by activating SIRT1 in hippocampus of rats北大核心CSCD

Aim To explore the inhibitory effect of Buyang Huanwu Decoction on the inflammatory response in the hippocampus of brain tissues of CIRI rats by regulating SIRT1 and the underlying mechanism. Methods The middle cerebral artery embolization (MCAO) model was prepared in rats and divided into sham operation group (Sham), model group (MCAO/R), Buyang Huanwu Decoction group (BYHWT),and BYHWT + SIRT1 inhibitor group (BYHWT + EX527). Zea Longa was used to detect the neurological function score of rats in each group; TTC staining was used to determine the volume of cerebral infarction; HE staining was used to observe the pathological damage of the hippocampus; Western blot was used to detect the expression levels of SIRT1 and IL-6; immunohistochemistry was used to detect TNF-α, IL-1β expression level. Results Compared with the sham group,the neurological function score of the MCAO/R group increased (P < 0.05); the volume of cerebral infarction increased (P < 0.05); the nerve cells in hippocampus were severely damaged, arranged disorderly, and the nucleus was broken; Western blot showed that the expression of SIRT1 decreased, IL-6 expression increased (P <0.05); immunohistochemistry showed that TNF-α,IL-1β expression increased (P < 0.05). Compared with the MCAO/R group, the neurological function score of the BYHWT group decreased (P <0.05); the volume of cerebral infarction decreased (P < 0.05); the damage of nerve cells in hippocampus was reduced; Western blot showed that the expression of SIRT1 increased and IL-6 expression decreased (P < 0.05); immunohistochemistry showed that TNF-α, IL-1β expression decreased (P < 0.05). Compared with the BYHWT group, the neurological function score of the BYHWT + EX527 group increased (P < 0.05); the volume of cerebral infarction was raised (P <0.05); the damage of nerve cells in hippocampus was aggravated; Western blot showed that the expression of SIRT1 decreased and IL-6 expression increased (P < 0.05); immunohistochemistry showed that TNF-α, IL-1β expression increased (P < 0.05). Conclusions Preliminary discussion of Buyang Huanwu Decoction can activate SIRT1 in hippocampus of rat brain tissues to reduce the inflammatory response after CIRI and play a role in brain protection. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Daidzein affects proliferation and apoptosis in non-small cell lung cancer cells:role of p53 signaling pathway北大核心CSCD

Aim To investigate the effects of daidzein（DD） on the proliferation and apoptosis of non-small cell lung cancer cells,with a focus on the possible role of the p53 signaling pathway in this regard. Methods CCK-8 method and flow cytometry were used to detect the effects of soy isoflavone crude extract and DD on the viability and apoptosis of HELF and H1299 cells. Gene microarray was used to detect the changes in gene expression after treatment of H1299 cells with DD. GSEA and differential analysis were used to screen the major pathways and key genes. RT-qPCR and Western blot were performed to verify the differences in mRNA and protein expression of key genes（p53 and CASP9） in the major pathways. After p53 inhibitor Pifithrin-α inhibited the expression of p53,the effect of DD on p53 mRNA and protein expression levels was examined,and the proliferative effect on H1299 cells was observed. Results Soy isoflavone crude extract and DD promoted proliferation and inhibited apoptosis of normal lung cells and inhibited proliferation and promoted apoptosis of lung cancer cells. p53 signaling pathway was significantly enriched in the DD-treated group（NES=1.78,P=0.000）,and the expressions of p53 and CASP9 genes were found to be significantly up-regulated in the treated group. Compared with the control group,mRNA expression of CASP9 and p53 significantly increased in both HELF and H1299 cells treated with DD（P<0.05）,and p53 protein expression also increased in HELF cells（P<0.05）. After inhibition of p53 expression,DD significantly increased the mRNA expression of p53 in H1299 and HELF cells（P<0.05） and also markedly increased the expression of p53 protein in H1299 cells（P<0.05）,and it was observed that DD inhibited the proliferation of lung cancer cells. Conclusions DD inhibits the proliferation and promotes the apoptosis of lung cancer H1299 cells,and the mechanism mainly involves the p53 signaling pathway. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Effect of corilagin on cholesterol metabolism in macrophages and its mechanism北大核心CSCD

Aim To elucidate the effect of corilagin (Cor) on cholesterol metabolism in macrophages and the underlying mechanism. Methods Molecular docking was applied to predict the protein target of Cor on cellular cholesterol metabolism. The RAW264.7 macrophage foam model induced by 80 mg • L-1 oxidized low density lipoprotein (ox-LDL) was established to evaluate the activity of Cor on lowering-cholesterol. The expression of genes and proteins related with cholesterol metabolism were detected by q-PCR and Western blotting,respectively. Then the activity of Cor on lipid metabolism was validated in ApoE mice fed with high-fat-diet. Results Cor and Class A Scavenger receptor (SRA), CD36, peroxisome proliferator-activated receptor γ (PPAR-γ), ATP binding cassette transporter Gl(ABCGl), which associated with cholesterol metabolism, could form hydrogen bonds and hydrophobic interactions. Cell experiments showed that Cor (60,120 and 240 μmol • L-1) significantly decreased TC content in macrophages, Cor could down-regulate SRA and CD36 gene expression, SRA protein expression, up-regulate the expression of ABCA1 and ABCG1 genes. Animal experiments demonstrated that Cor (15,30 and 60 mg • k g - 1 ) could decrease the serum TMAO content, the plaque area and formation of foam cells in the aortic root,the expression levels of CD36 and SRA fluorescent proteins in aortic root plaques. Conclusions Cor could inhibit the formation of macrophage foam cells through the regulation of cholesterol metabolism mediated by CD36,SRA, ABCA1 and ABCG1 to cure the AS. © 2023 Publication Centre of Anhui Medical University. All rights reserved.