Effect and mechanism of adipocyte co-culture on aquaporin-9 expression in HepG2 cells / 中华肝脏病杂志

Objective@#To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action.@*Methods@#Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples.@*Results@#The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001).@*Conclusion@#Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

Recombinant human granulocyte-colony-stimulating factor increases the sensitivity of acute myeloid leukemia cells to arsenic trioxide and its possible mechanism / 肿瘤

Objective: To investigate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) combined with arsenic trioxide (ATO) (molecular formula: As2O3) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells, and to explore the possible mechanisms. Methods: AML cells HL-60 and THP-1 were pre-treated with rhG-CSF (100 ng/mL), and then treated with different concentrations of As2O3. The relative proliferation rate was detected by CCK-8 method, while the apoptosis and cell cycle distribution were measured by FCM method. The expression levels of aquaporin 9 (AQP9) mRNA and protein in HL-60, THP-1 and acute promyelocytic leukemia NB4 cells as well as HL-60 and THP-1 cells treated with rhG-CSF (100 ng/ mL) were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: RhG-CSF promoted the proliferation of HL-60 and THP-1 cells (both P < 0.01). Compared with the As2O3 group, rhG-CSF pre-treatment combined with 2 μmol/L As2O3 inhibited the proliferation of HL-60 and THP-1 cells (both P < 0.05), rhG-CSF combined with different concentrations of As2O3 increased the apoptotic rates of HL-60 and THP-1 cells (both P < 0.05). As2O3 caused G0/G1 arrest in HL-60 and THP-1 cells (both P < 0.05). rhG-CSF caused S-phase arrest in HL-60 and THP-1 cells (both P < 0.01), the effect was more obvious in rhG-CSF combined with As2O3 group (both P < 0.05). The expressions of AQP9 mRNA and protein in HL-60 and THP-1 cells were lower than those in NB4 cells (all P < 0.01). Compared with the untreated control group, 100 ng/mL rhG-CSF up-regulated the expression levels of AQP9 mRNA and protein in HL-60 and THP-1 cells (all P < 0.05). Conclusion: RhG-CSF can increase the sensitivity of non-M3 AML cells to As2O3, which may be associated with the up-regulation of AQP9 expression.

Aquaporin 9 and tumor / 国际肿瘤学杂志

Aquaporin 9 (AQP9),one of members of the cell membrane protein family,plays an important role in maintaining metabolism and water balance in vivo.AQP9 could also play an importan role in development of tumors,such as migration,metastasis and apoptosis of tumor cells.In addition,AQP9 is of great significance in judging prognosis of tumors.

Overexpression of AQP9 inhibits the proliferation of human hepatocarcinoma SMMC-7721 and Huh-7 cells in hypoxic microenvironment / 肿瘤

Objective: To investigate the aquaporin 9 (AQP9) involved in the hypoxia response of hepatocellular carcinoma cells in hypoxic microenvironment, and to explore its possible mechanism. Methods: The hypoxia model of hepatocellular carcinoma SMMC-7721 and Huh-7 cells was constructed by using hypoxia incubator. The expression of AQP9 mRNA in SMMC-7721 and Huh-7 cells and the expression of AQP9 protein in Huh-7 cells after hypoxic incubation were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After SMMC-7721 and Huh-7 cells were infected with recombinant lentivirus LV-AQP9 with AQP9 over-expression or empty vector lentivirus LV-PWPI, the SMMC-7721-AQP9 and Huh-7-AQP9 cells with stable over-expression of AQP9 as well as the negative control SMMC-7721-PWPI and Huh-7-PWPI cells were established. The proliferation and the expressions of hypoxia-inducible factor-1α (HIF-1α), mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) in SMMC-7721-AQP9, SMMC-7721-PWPI, Huh-7-AQP9 and Huh-7-PWPI cells after hypoxic incubation were detected by CCK-8 assay and Western blotting, respectively. Results: In hypoxic microenvironment, the expression levels of AQP9 mRNA and protein in Huh-7 cells were decreased (both P < 0.05), and the expression level of AQP9 mRNA in SMMC-7721 cells was decreased (P < 0.05). As compared with normoxic microenvironment, the inhibitory effect of over-expression of AQP9 on the proliferation of SMMC-7721 and Huh-7 cells was stronger in hypoxic microenvironment (both P < 0.05). The expression levels of HIF-1α and p-mTOR proteins in SMMC-7721-AQP9 and Huh-7-AQP9 cells after hypoxic incubation were significantly lower than those in the SMMC-7721-PWPI and Huh-7-PWPI cells (all P < 0.05). Conclusion: In hypoxic microenvironment, the over-expression of AQP9 may inhibit the proliferation of SMMC-7721 and Huh-7 cells by down-regulating the expression of HIF-1α, and this inhibitory effect is stronger than in normoxic microenvironment.

Research on the expression of aquaporin 9 gene and efficacy of mannitol in neonatal rats with hypoxia-ischemic brain edema / 中国新生儿科杂志

Objective To study the relationship between aquaporin 9 (AQP 9) gene and brain edema in neonatal rats of hypoxic-ischemic brain damage (HIBD) and the therapeutic mechanism of mannitol.Method Healthy and 7-day-old SD rats were randomly assigned into three groups:sham-operated group,HIBD group and mannitol group.Both HIBD and mannitol group were established on HIBD model.The mannitol group was given mannitol intraperitoneally at 0,24,48 h of HIBD.2 ml/kg of 2％ Evans blue (EB) were injected intraperitoneally before sacrifice.0,6,12,24,48 and 72 h after HIBD,the outcomes were analyzed including the brain water content,the expression of AQP 9 mRNA measured using RT-PCR and immunofluorescence staining methods,and the permeability of blood-brain barrier (BBB) measured with EB.Result In HIBD group,the brain water content was higher comparing with sham-operated group at 0 h after HIBD(P ＜ 0.05),and gradually increased over time,reaching peak at 48 h (89.3％ ± 1.9％) and then decreased.In mannitol group,brain water content started to decrease from 1 h after mannitol administration to the bottom at 12 h (86.5％ ±0.6％),then increased to peak at 72 h (87.2％ ± 1.7％),and brain water content were decreased during 0 ～ 48 h comparing with HIBD group.HIBD group's EB were higher than sham-operated group (P ＜ 0.05);Mannitol group's EB were decreased comparing with HIBD group (except 0 h,P ＜ 0.05).AQP 9 mRNA expression in the HIBD group was decreased at 0 h,and reached the bottom at 48 h (0.09 ± 0.07).Comparing with sham-operated group,it was higher in the HIBD group at0,6,72 h,and lower (P＜ 0.05) at 12,24,48 h.Higher AQP 9 mRNA expression were detected in mannitol group than HIBD group and sham-operated group at each time point (with the exception of 48 h) (P ＜ 0.05).AQP 9,which was closely related to water metabolism,were widely found in the pia mater and ependyma using immunofluorescence staining.After ischemia and hypoxia insult,an increasedecrease-increase pattern of AQP 9 expression was found.Conclusion AQP 9 is widely existed in various parts of the brain,influencing brain edema through a variety of pathways.AQP 9 also plays a role in alleviating brain edema in mannitol therapy.

Effect ofβ-sodium Aescinate on the Expression of Aquaporin-4 and Aquaporin-9 in Rats with Spinal Cord Injury / 中国医科大学学报

Objective To study the effect ofβ?sodium aescinate on the expressions of aquaporin?4 and aquaporin?9 in rats with spinal cord inju?ry. Methods A total of 150 rats were randomly divided into 3 groups:sham group(n=50),spinal cord injury(SCI)group(n=50)andβ?sodi?um aescinate group(n=50). The experimental animal models was established by modified Allen’s model. The Basso,Beattie and Bresnahan (BBB)locomotor rating scale and inclined plane test were used to evaluate rat behavioral consequences after injury.The immunohistochemical staining and western blotting assay were performed to observe the expressions of aquaporin?4 and aquaporin?9. Results Compared with sham group,BBB score and inclined plane test score of SCI group andβ?sodium aescinate group were significantly lower at each time point(P<0.05);however,the functional recovery was significantly better inβ?sodium aescinate group than in SCI group at each time point from 7 d after SCI(P<0.05). The aquaporin?4 and aquaporin?9 positive expressions of rats in sham group were lower significantly than rats in SCI group andβ?sodium aescinate group(P<0.05);however,the aquaporin 4 and aquaporin 9 positive expressions of rats inβ?sodium aescinate group was lower signifi?cantly than rats in SCI group at each time point(P<0.05). Conclusion β?sodium aescinate can protect the neurologic function in rats with spi?nal cord injury by decreasing aquaporin?4 and aquaporin?9 protein expression.

Inhibiting effect of AQP9 over-expression on the growth of hepatoma carcinoma SMMC-7721 cells subcutaneously transplanted in nude mice and its possible mechanism / 肿瘤

Objective: To investigate the effect of aquaporin 9 (AQP9): on the growth of human hepatoma carcinoma SMMC-7721 cells in nude mice and its possible mechanism. Methods: SMMC-7721 cells were infected with recombinant lentivirus LV-AQP9 (empty lentivirus LV-CFP as a negative control). The infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was observed under a laser scanning confocal microscope, and the expression levels of AQP9 mRNA and protein were detected by real-time fluorescence-based quantitative PCR and Western blotting, respectively. The apoptosis of SMMC-7721 cells was analyzed by FCM and 4, 6-diamidino-2-phenylindole (DAPI): staining, respectively. The proliferation ability of SMMC-7721 cells was analyzed by cell counting kit-8 (CCK-8). Recombinant hepatoma carcinoma SMMC-7721/LV-AQP9 (AQP9 group): and SMMC-7721/LVCFP cells (CFP group): were subcutaneously implanted in nude mice. The volume and growth rate of the xenograft tumors were observed. Pathological alteration in xenograft tumors and lung tissues were observed by HE-staining. The expression level of proliferating cell nuclear antigen (PCNA): protein was detected by immunohistochemical staining. Results: The infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was about 90% under a laser scanning confocal microscope. The AQP9 mRNA and protein expression levels in SMMC-7721 cells were significantly increased (both P < 0.01). AQP9 over-expression could significantly promote the apoptosis of SMMC-7721 cells (P < 0.05), and significantly inhibit the proliferation of SMMC-7721 cells at different time points (48-96 h): after transfection (all P < 0.05). The results of ANOVA for repeated measurement of xenograft tumor volume displayed that xenograft tumor volume of AQP9 over-expression group was smaller than that of CFP group, and the growth rate was slower (Fgroup = 79.161, Pgroup = 0.000; Ftime = 101.965, Ptime, = 0.000; Fgroupxtime = 18.481, Fgroupxtime = 0.002). Immunohistochemistry staining showed that the expression level of PCNA protein in the xenograft tumor of over-expression group significantly decreased compared with that of the control group. Conclusion: AQP9 over-expression can inhibit the growth of xenografts of human hepatoma carcinoma SMMC-7721 cells in nude mice and possibly conduct dual control by promoting apoptosis and inhibiting the proliferation, which provides the foundation for further research on molecular mechanism, and it is expected to become a new target for cancer treatment.

Aquaporin 9 in rat brain after severe traumatic brain injury / Aquaporina 9 em cérebro de rato após traumatismo cerebral grave

OBJECTIVE: To reveal the expression and possible roles of aquaporin 9 (AQP9) in rat brain, after severe traumatic brain injury (TBI). METHODS: Brain water content (BWC), tetrazolium chloride staining, Evans blue staining, immunohistochemistry (IHC), immunofluorescence (IF), western blot, and real-time polymerase chain reaction were used. RESULTS: The BWC reached the first and second (highest) peaks at 6 and 72 hours, and the blood brain barrier (BBB) was severely destroyed at six hours after the TBI. The worst brain ischemia occurred at 72 hours after TBI. Widespread AQP9-positive astrocytes and neurons in the hypothalamus were detected by means of IHC and IF after TBI. The abundance of AQP9 and its mRNA increased after TBI and reached two peaks at 6 and 72 hours, respectively, after TBI. CONCLUSIONS: Increased AQP9 might contribute to clearance of excess water and lactate in the early stage of TBI. Widespread AQP9-positive astrocytes might help lactate move into neurons and result in cellular brain edema in the later stage of TBI. AQP9-positive neurons suggest that AQP9 plays a role in energy balance after TBI.

OBJETIVO: Revelar a expressão e os possíveis papéis da aquaporina 9 (AQP9) no cérebro de ratos após lesão cerebral traumática (LCT) grave. MÉTODOS: Foram utilizados: determinação do conteúdo cerebral de água, corante cloreto de tetrazólio, corante azul de Evans, imunoistoquímica (IHQ), imunofluorescência (IF), western blot e PCR em tempo real. RESULTADOS: O conteúdo cerebral de água alcançou o primeiro e o segundo (o mais alto) picos após 6 e 72 horas. A função da barreira hematoencefálica se mostrou muito prejudicada após 6 horas da LCT. A pior isquemia cerebral ocorreu após 72 horas da LCT. Astrócitos AQP9 positivos e neurônios no hipotálamo foram detectados difusamente pela IHQ e IF após LCT. A abundância de AQP9 e de sua mRNA aumentou após LCT e alcançou dois picos após 6 e 72 horas, respectivamente, da LCT. CONCLUSÕES: AQP9 aumentada pode contribuir para a eliminação de água e lactato em excesso na fase precoce da LCT. Astrócitos difusamente localizados AQP9 positivos podem ajudar a entrada do lactato nos neurônios, promovendo edema cerebral celular na fase tardia da LCT. Neurônios AQP9 positivos sugerem que AQP9 tem um papel no equilíbrio energético após LCT.

Enhanced Expression of Aquaporin-9 in Rat Brain Edema Induced by Bacterial Lipopolysaccharides / 华中科技大学学报（医学）（英德文版）

To investigate the role of AQP9 in brain edema,the expression of AQP9 in an infectious rat brain edema model induced by the injection of lipopolysaccharide (LPS) was examined.Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expressions of AQP9 mRNA and protein at all observed intervals were significantly increased in LPS-treated animals in comparison with the control animals.Time-course analysis showed that the first signs of blood-brain barrier disruption and the increase of brain water content in LPS-treated animals were evident 6 h after LPS injection,with maximum value appearing at 12 h,which coincided with the expression profiles of AQP9 mRNA and protein in LPS-treated animals.The further correlation analysis revealed strong positive correlations among the brain water content,the disruption of the blood-brain barrier and the enhanced expressions of AQP9 mRNA and protein in LPS-treated animals.These results suggested that the regulation of AQP9 expression may play important roles in water movement and in brain metabolic homeostasis associated with the pathophysiology of brain edema induced by LPS injection.

Aquaporin-1 and Aquaporin-9 Expressed by Reactive Astrocytes after Traumatic Brain Injury in Rat / 中国康复理论与实践

@#Objective To investigate the expression changes of aquaporin-1 (AQP1) and aquaporin-9 (AQP9) in rats after traumatic brain injury, and explore the roles of AQP1 and AQP9 in the development of brain edema.Methods In an impact-acceleration head injury model of rat, brain water content was measured by wet-dry weight method at 1 h, 6 h, 24 h, 48 h and 168 h after brain trauma. The expressions of AQP1 and AQP9 in brains were investigated by immunohistochemistry and Western blot.Results Brain water content increased in the injuried brain tissues after 1 h post-injury, reached peak at 24 h and returned at 168 h. Both expressions of AQP1 and AQP9 were induced in reactive astrocytes adjacent to injury sit at 6 h and 24 h after brain trauma. Staining of AQP1 in the endothelial cells was not present in normal rat brains but appeared strong after brain trauma.Conclusion A remarkable induction expression of AQP1 or AQP9 after brain trauma may participate in the development of brain edema.

Effects of pyrrolidine dithiocarbamate on aquaporin-9 expression and brain edema around the hematoma in experimental intracerebral hemorrhage / 临床神经病学杂志

Objective To investigate the effects of pyrrolidine dithiocarbamate(PDTC) on aquaporin-9(AQP9) expression and brain edema around the hematoma in experimental intracerebral hemorrhage(ICH).Methods 128 SD rats were randomly divided into four groups: normal group,control group,ICH group and PDTC group.According to Rosenberg's methods,the models of experimental ICH were established.The rats in PDTC group were received PDTC intraperitoneally 2 h later.The brain weight column(BWC) at different time point was calculated by gray-wet method,and the expression of AQP9 was examined by immunohistochemistry.Results The BWC and expression of AQP9 in hematoma side of ICH begin to increase 4 h after ICH,peaked at 72 h,and decreased 120 h later.The BWC in opposite side of hematoma also increased 4 h after ICH and peaked at 72 h.Compared with control group,the BWC and expression of AQP9 in ICH group were higher,especially in hematoma side of brain(all(P

Changes and their relationship of aquaporin-9 mRNA expression and Ca~(2+) concentration after cerebral ischemic in rats / 临床神经病学杂志

Objective To investigate the changes and their relationship of aquaporin-9(AQP9) mRNA expression and Ca2+ concentration of brain tissue after cerebral ischemic.Methods The models of cerebral ischemic in the rats were made by occluding unilateral middle cerebral artery with the suture method.The expression level of AQP9 mRNA was assessed by RT-PCR at interval times of 6 h,1 d,2 d,3 d,5 d after cerebral ischemic,respectively.Fura-2/AM fluoremetry technique was used to determine the cellular Ca2+ concentration of brain tissue.The results were compared with control group.Results Compared with control group,the expression level of AQP9 mRNA and the concentrations of Ca2+ significantly increased at 6 h in ischemic edema tissue,and reached a peak at 2 d,3 d after cerebral ischemic(P

AQP9 expression in rat cerebral cortex and hippocampus after moderate traumatic brain injury / 第三军医大学学报

Objective To observe the expressions of AQP9 and AQP9 mRNA in cerebral cortex and hippocampus of rats after moderate traumatic brain injury(TBI).Methods The moderate TBI model was established according to Feeney's method.At different time points after TBI,the degree of cerebral ischemia by TTC staining,the expressions of AQP9 and its mRNA in brain by immunohistochemistry and RT-PCR respectively were performed.Results No obvious ischemia in brain was found 6 h after TBI,but gradually the ischemic focus enlarged,and reached the maximum 72 h after TBI.The expressions of AQP9 and its mRNA were similar,increasing significantly 3 h after TBI,decreasing 6 h after TBI,then increasing again at 24 h in bilateral cerebral cortex and the hippocampus and reaching the maximum 48 h or 72 h after TBI.Conclusion The increased expression of AQP9 after TBI is involved in the stress of early TBI and in later brain energy metabolism.