Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.@*METHODS@#Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.@*RESULTS@#Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).@*CONCLUSION@#Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo.@*METHODS@#Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining.@*RESULTS@#Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01).@*CONCLUSION@#EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

Mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein in Chlamydia trachomatis via high-mobility group box 1 protein: a preliminary study / 中华皮肤科杂志

Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis,so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis.Methods HeLa cells were divided into two groups:carbonyl cyanide m-chlorophenyl hydrazone (CCCP,an apoptosis inducer) group was stimulated by CCCP for 30 minutes,and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes.Then,Western blot analysis was performed to determine the expression of apoptosisrelated proteins Bcl-2,Bax and caspase-3,JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells,and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay.To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein,HMGB 1 shRNA and control RNA were separately transfected into the HeLa cells,which were then stimulated by pORF5 plasmid protein and CCCP.Then,the protein expression of Bcl-2,Bax,activated caspase-3 was determined,and cytochrome c release was analyzed.Data were compared between two groups by using paired t test.Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential,and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3;t =6.95,P ＜ 0.01).The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t =8.62,P ＜ 0.01),while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79％ ± 10％ (t =9.23,P ＜ 0.01) and 75％ ± 8％ (t =4.26,P ＜ 0.05) respectively compared with the CCCP group.Compared with the control RNA transfection group,the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t =11.23,P ＜ 0.01),increased cytochrome c release,decreased Bcl-2 expresson (t =7.19,P ＜ 0.05) and increased Bax expression (t =13.06,P ＜ 0.01) after stimulation with pORF5 and CCCP.Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.

Effects of Different Types of Electroacupuncture on Expression of High Mobility Group Box Protein-1 and Interleukin-10 in Hippocampus of APP/PS1 Mice / 中国康复理论与实践

@#Objective To compare the effect of impulse electroacupuncture (impulse-EA) and music electroacupuncture (music-EA) for Tongdu Qishen on spatial learning and memory, and expression of cytokine in the hippocampus of APP/PS1 mice. Methods A total of 32 APP/PS1 double transgenic male mice were randomly divided into model group (n=8), drug group (n=8), impulse-EA group (n=8) and music-EA group (n=8), the same background and age male C57BL/6 mice were observed as normal group (n=8). The impulse-EA group and music-EA group accepted EA at Baihui (GV20) and Yintang (GV29), connected with their own electroacupuncture stimulators, for 20 minuts, then, they were pricked Renzhong (GV26) for a while. The drug group accepted donepezil hydrochloride 0.92 mg/kg intra-gastrically. The normal group, model group and drug group were grabbed and bounded in the same way. After 15 days of treatment, they were assessed with Morris water maze. The expression of high mobility group box pro-tein-1 (HMGB1) and interleukin-10 (IL-10) in hippocampus were measured with immunohistochemistry and Western blotting. Results The escape latency shortened inEA groups compared with that of the model group since the fourth day of Morris water maze training (P<0.05), and it was the least in the music-EA group; and the ratio of swimming across the target quadrant increased (P<0.05), but there was no significant difference between EA groups (P>0.05). The ex-pression of HMGB1 decreased (P<0.05) and the expression of IL-10 increased (P<0.05) in EA groups com-pared with that of the model group, and HMGB1 decreased more and IL-10 increased more in the music-EA group than in the impulse-EA group (P<0.05), according to the measurement in immunohistochemistry, not in Western blotting. Conclusion Both impulse-EA and music-EA based on Tongdu Qishen can promote the recovery of the learning and memory in APP/PS1 mice, and music-EA may do more effectively in inhibition of pro-inflammatory factors and promotion of the anti-inflammatory factors.

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1,NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis / 中国病理生理杂志

AIM:To observe the effect of Yiqi Huayu Huatan decoction(YHHD)on unilaterral ureteral ob-struction(UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism.METHODS: Fe-male SD rats(n=48)were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups,with 8 rats in each group.The UUO model rats was established by ligating left ureter.The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily.After 12 weeks,the rats were sacrificed.The serum samples were collected for determining the concentrations of cystatin C(Cys-C)and uric acid(UA).The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining.The mRNA expression of Krüppel-like factor 15(KLF15),high-mo-bility group box protein 1(HMGB1),nuclear factor-κB(NF-κB),IκB,monocyte chemotactic protein-1(MCP-1),inter-leukin-1β(IL-1β), tumor necrosis factor-α(TNF-α),fibronectin(FN),collagen type I(Col I)and Col-Ⅳwas detec-ted by real-time PCR.The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot.The protein expression of MCP-1 was determined by the method of immunohistochemistry.RESULTS:Compared with sham group,the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased(P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated(P<0.05), while the mRNA expression of HMGB1,NF-κB,IκB,MCP-1,IL-1β,TNF-α,FN,Col I and Col Ⅳand the protein expression of HMGB1,NF-κB and MCP-1 were significantly up-regulated(P<0.05).Compared with model group,the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased(P<0.05),with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1βand TNF-α(P<0.05).The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group(P<0.05),while the protein ex-pression of MCP-1 and the mRNA expression of FN were significantly down-regulated(P<0.05).The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group(P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated(P<0.05).The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group(P<0.05).No significant difference of UA level among the groups was observed.CONCLUSION:YHHD allevi-ates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect.The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its down-stream inflammation-related factors in the renal tissue.

Clonidine inhibits inflammatory response in lung injury mice through cholinergic anti-inflammatory pathway / 中国病理生理杂志

AIM: To explore the influence of clonidine on inflammatory response in lung injury mice and its possible mechanism.METHODS: Clonidine solution was intravenously injected into the mice with lung injury induced by LPS.The left upper lobe of the lung was collected to detect lung wet/dry weight ratio (W/D) and total lung water content (TLW).The concentrations of IL-6, IL-1β and TNF-α were measured by ELISA.The expression of α7 nicotinic acetylcholine receptor (α7nAChR) and high-mobility group box protein 1 (HMGB1) at mRNA and protein levels was determined by RT-PCR and Western blot.After importing α7nAChR siRNA lentiviral vector or injecting exogenous HMGB1 protein, the inflammatory cytokines were detected.RESULTS: Clonidine attenuated lung injury and inhibited inflammatory reaction.Clonidine promoted the activation of cholinergic anti-inflammatory pathway by promoting α7nAChR expression.Clonidine inhibited HMGB1 expression, which promoted the secretion of IL-6, IL-1β and TNF-α.HMGB1 was negatively regulated by α7nAChR.CONCLUSION: Clonidine functions as an anti-inflammatory reagent to the lung injury mice.The mechanism may be related to activating the cholinergic anti-inflammatory pathway and inhibiting the expression of HMGB1.

Expression and significance of HMGB1 and RAGE in patients with lupus nephritis / 安徽医科大学学报

Objective To explore the role of high mobility group box protein 1 ( HMGB1 ) and receptor for ad-vanced glycation endproducts( RAGE) in lupus nephritis( LN) . Methods The serum and urine levels of HMGB1 were detected by ELISA in SLE patients and normal controls, and intrarenal expressions of HMGB1 and RAGE were detected by immunohistochemistry in renal tissues of SLE patients and normal-appearing renal tissues. Results The serum and urine levels of HMGB1 were significantly higher in SLE patients compared to healthy controls( P<0. 01 ) , in patients with active disease compared to those with inactive disease ( P <0. 05 ) , serum levels of HMGB1 were found to be significantly higher in patients with renal involvement compared to those without renal in-volvement(P<0. 05). In addition, the levels of serum HMGB1 showed positive correlation with SLEDAI,urine pro-tein(24 h) and lencocyte count (P <0. 05). The expression of urine HMGB1 showed positive correlation with SLEDAI ( P<0. 05 ) . Intermediate-intensity staining of HMGB1 and RAGE was detected in renal tubules in normal-appearing renal tissues, however, both renal tubules and glomerular cells had the strong expression of HMGB1 and RAGE in SLE patients. The intrarenal production of HMGB1 and RAGE in SLE patients was obviously higher than that in normal-appearing renal tissues(P<0. 05), and the expression levels of HMGB1 in SLE IV and IV+V were higher than those in SLE II ( P<0. 05 ) . The expression levels of RAGE in SLE IV were higher than those in SLE II(P<0. 05). Conclusion HMGB1 and RAGE may play key roles in the pathogenesis of LN and may associate with the pathology category of LN.

The relationship between the ultrasonic sign and expression of HMGB 1,VEGF in serum of hepatocellular carcinoma / 重庆医学

Objective To investigate the relationship between the ultrasonic sign and expression of HMGB1 ,VEGF in serum of hepatocellular carcinoma patients .Methods Doppler ultrasound was used to inspect the size of the tumor and to analyze the typing and flow classification .The ELISA was used to check the HMGB1 and VEGF in serum .Results The expression level of HMGB1 in nodular and massive group was higher than that in small modelgroup (53 .5 ± 4 .3) ng/mL .The expression level of HMGB1 in massive group(102 .9 ± 9 .7)ng/mL was higher than that in nodular group(76 .0 ± 6 .2) ng/mL(P0 .05) .With the increase of flow classification ,the levels of HMGB1 and VEGF also increased(P<0 .05) .Conclusion The HMGB1 in serum has some connection with morphology types .The HMGB1 and VEGF in serum increase with the flow classification .

Effect of curcumin on learning-memory ability and expression of HMGB1 and JNK in rat model of Alzheimer disease / 中国病理生理杂志

AIM:To evaluate the effect of curcumin on impaired learning-memory ability and the expression of high mobility group box protein 1 ( HMGB1 ) and c-Jun N-terminal kinase ( JNK ) in a rat model of Alzheimer disease (AD).METHODS:Male Sprague-Dawley rats, weighing 250~270 g, were randomly divided into 4 groups (n=9):blank control group (group A), model group (group B), curcumin treatment group (group C, curcumin injected intraper-itoneally at 100 mg· kg-1· d-1 for 6 consecutive days) and solvent control group (group D).The rats of AD model were induced by injection of ibotenic acid into the nucleus basalis of Meynert ( NBM) bilaterally.All rats were trained in Morris maze to assess the ability of learning and memory .The expression of HMGB1 and JNK in the hippocampus was detected by the methods of immunohistochemistry and Western blotting .RESULTS:Compared with group A , the average escape laten-cy (AEL) in groups B and D were obviously longer (P0.05).No significant difference in the expression of HMGB1 in the hippocampus among the 4 groups was found (P>0.05).However, compared with groups B and D , the expression of JNK in group C was decreased obvi-ously (P<0.05).CONCLUSION: Curcumin significantly improves the learning and memory ability of AD rats .The probable mechanisms may be related to inhibiting the release of HMGB 1 from the nucleus of hippocampal neurons and de-creasing the expression of JNK in the hippocampus .

Screening of binding proteins of HMGB1 promoter by phage display technique / 中国病理生理杂志

AIM: To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS: HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme-linked immunosorbent assay (ELISA). Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS: Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein-1(AP-1) and two of which were uncharacterized ones. CONCLUSION: Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.