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Objective:To evaluate the relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and postsynaptic density protein-95 (PSD-95)/Kalirin-7/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway in neonatal rats.Methods:Sixty SPF male Wistar rats, aged 7 days, weighing 12-18 g, were divided into 5 groups ( n=12 each) using a random number table method: control group (group C), 1% sevoflurane anesthesia for 2 h group (group S 1), 1% sevoflurane anesthesia for 4 h group (group S 2), 2% sevoflurane anesthesia for 2 h group (group S 3) and 2% sevoflurane anesthesia for 4 h group (group S 4). Morris water maze test was performed at 4, 8 and 12 weeks after anesthesia.The rats were sacrificed after the last Morris water maze test, and the hippocampal tissues were obtained for microscopic examination of the pathological changes (using HE staining), neuron apoptosis (by TUNEL staining), and expression of PSD-95, Kalirin-7 and Rac1 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction). The apoptosis rate was calculated. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the time of stay in the target quadrant was shortened, and the apoptosis rate of hippocampal neurons was increased at 4th, 8th and 12th weeks after anesthesia, phosphorylated Rac1/Rac1 ratio was decreased, and the expression of PSD-95 and Kalirin-7 protein and mRNA was down-regulated in S 1, S 2, S 3 and S 4 groups ( P<0.05). Compared with group S 4, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of stay in the target quadrant was prolonged, and the apoptosis rate of hippocampal neurons was decreased, phosphorylated Rac1/Rac1 ratio was increased, the expression of PSD-95 and Kalirin-7 protein and mRNA was up-regulated, and the histopathological changes of hippocampal tissues were attenuated in S 1, S 2 and S 3 groups ( P<0.05). Conclusions:The mechanism by which sevoflurane anesthesia induces long-term learning and memory impairment may be related to inhibition of activity of PSD-95/Kalirin-7/Rac1 signaling pathway in hippocampi of neonatal rats.
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Objective To evaluate the effect of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes after the plasmid containing the Wnt5A gene is transfected into primary melanocytes.Methods In vitro cultured primary human melanocytes were divided into three groups:blank control group receiving no treatment,negative control group transfected with endotoxin-free pcDNA3.1 (+)empty vector by Lipo3000 in Opti-MEM medium,Wnt5A plasnid group transfected with endotoxin-free pcDNA3.1 (+) vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium.After the transfection,quantitative PCR (qPCR) was performed to measure the mRNA expression of Wnt5A,ras-related C3 botulinum toxin substrate 1 (Rac1),filamentous actin (F-actin) and β-tubulin,Western blot analysis to determine the protein expression of Wnt5A,receptor tyrosine kinase like orphan receptor 2 (ROR2),Rac1,F-actin and β-tubulin,and an immunofluorescence assay (IFA) to observe the expression of cytoskeletal proteins.Results qPCR showed significant differences in the mRNA expression of the Wnt5A gene and its downstream genes Rac1 and F-actin among the Wnt5A plasmid group,negative control group and blank control group (F =1374.179,112.576,66.458,respectively,all P ＜ 0.01),but there was no significant difference in the mRNA expression of β-tubulin among the three groups (P ＞ 0.05).Additionally,the Wnt5A plasmid group showed significantly higher mRNA expression of Wnt5A,Rac1 and F-actin compared with the blank control group and negative control group (all P ＜ 0.05).As Western blot analysis revealed,compared with the blank control group and negative control group,the Wnt5A plasmid group showed significantly higher Wnt5A protein expression (both P ＜ 0.05),but significantly lower protein expression of Rac 1,ROR2 and F-actin (all P ＜ 0.05).However,no significant difference in β-tubulin protein expression was observed among the three groups (P ＞ 0.05).IFA showed no obvious difference in the fluorescence intensity of β-tubulin or F-actin between the Wnt5A group and the two control groups,but melanocytes showed larger size and increased number of dendrites,and the cytoskeleton changed dramatically with varying fluorescence intensity of F-actin,fuzzy texture,fractured or locally clustered tonofilaments in the Wnt5A group.Conclusion The overexpression of the Wnt5A gene in melanocytes can regulate the mRNA and protein expression of cytoskeletal proteins,nake melanocytes larger and more dendritic,and cause changes in the cytoskeleton,which may facilitate the transportation of melanosomes,and participate in the occurrence of hyperpigmented diseases.
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Objective To explore the expression of Ras-related protein 11(Rab11)in hypoxia, the effect of Rab11 on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. Methods SiHa cells were divided into 4 groups, the normoxic blank group (normal culture in normoxia), the hypoxic blank group (normal culture in hypoxia), the negative control group [transfection of negative control small interfering RNA(siRNA)in hypoxia], the Rab11-siRNA group (transfection of Rab11 siRNA in hypoxia). Western blot was used to examine the expression of Rab11, integrin α5, integrin β3, phosphorylated focal adhesion kinase(p-FAK), phosphorylated phosphatidylinositol 3 kinase(p-PI3K) protein, together with the expression of Ras correlative C3 creotoxin substrate 1(Rac1), which was critical in regulating cell invasion. The mRNA expression of Rab11 in the 4 groups was detected by realtime-qPCR. The cell invasion was detected by matrigel assay, while the cell migration was detected by transwell assay. Immunofluorescence was used to identify intracellular location of Rac1 in SiHa cell. Results (1) The expression of Rab11, intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 in the normoxic blank group were 0.56±0.04, 0.33±0.03, 0.32±0.03, 0.36±0.03, 0.35±0.03 and 0.47±0.03, respectively. In the hypoxic blank group, they were 0.73±0.03, 0.74±0.03, 0.61±0.03, 0.62±0.03, 0.60±0.03 and 0.73±0.03, respectively. In the negative control group, their expressions were 0.72±0.03, 0.73±0.03, 0.59±0.03, 0.61±0.03, 0.59±0.03 and 0.72±0.03, respectively. While in the Rab11-siRNA group, they were 0.44±0.03, 0.30±0.03, 0.29±0.03, 0.30±0.03, 0.30±0.03 and 0.34±0.04, respectively. The expressions of Rab11, α5, β3, p-FAK, p-PI3K and Rac1 were significantly higher in the hypoxic blank group than in the normoxic blank group(P<0.05), and were significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group(P<0.05). (2) The expressions of Rab11-mRNA were 1.000±0.000, 1.454±0.114, 1.442±0.101, 0.570± 0.046 in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11- siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group(P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group(P<0.05). (3) By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab11-siRNA group were 65±12, 106±16, 104± 17 and 50±11, respectively. The invasion capacity was significantly higher in the hypoxic blank group than in the normoxic blank group(P<0.05), and was significantly lower in the Rab11- siRNA group than in the hypoxic blank group and the negative control group(P<0.05). (4) By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group were 127±12, 169±15, 161±13 and 77±13, respectively. The capacity of invasion was significantly higher in the hypoxic blank group than in the normoxic blank group(P<0.05), and was significantly lower in the Rab11- siRNA group than in the hypoxic blank group and the negative control group(P<0.05). (5) The immunofluorescence showed that the red fluorescence intensity around nucleus was significantly increased in the normoxic blank group, the hypoxic blank group and the negative control group than in the Rab11- siRNA group. Conclusions Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rab11 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 protein.
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Objective To explore the expression and significance of Rac1 and WAVE2 protein in glomerulus of high- fat diet induced C57BL/6J model mice. Methods Thirty-two male C57BL/6J mice (3-week old) were randomly assigned into two groups(16 in each group). The control group was fed with basic diet (10%fat) for 4 weeks. The high-fat diet group was fed with high-fat diet (60%fat) for 4 weeks. The kidney morphological changes were examined by HE and PAS staining. The expressions of Rac1 and WAVE2 protein were examined by Western blot and immunohistochemistry analysis. Results HE and PAS results showed that there were glomeruli mesangial matrix hyperplasia and exudation in high-fat diet group compared with control group. The immunohistochemistry and Western blotting results showed that expressions of Rac 1 and WAVE2 in glomerulus were both increased in high-fat diet group compared with those of control group. Conclusion Rac1 and WAVE2 protein may be involved in glomerular injuries induced by high-fat diet.
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Objective To investigate the expression of Rac 1 in the guinea pig cochlea of gentamicin-induced ototoxic damage and prevention with antioxidant sodium salicylate , and to explore the roles of Rac 1 in the ototoxic mechanism of aminoglycoside antibi-otics.Methods Thirty healthy male guinea pigs were involved in this study .All the guinea pigs were randomly divided into 3 groups and received intraperitoneal injections according to their arranged group .Group I (control) was treated with normal saline for 7 days. Group II [Gentamicin (GM)] was treated with gentamicin alone for 7 days.Group III [GM+sodium salicylate(SA)] was treated with gentamicin in combination with sodium salicylate for 7 days.Paraffin-embedded cochlear section with immunohistochemical stai-ning was used for evaluation of Rac 1 expression in the cochlea .The protein was extracted from the cochlea tissues , and Rac1 protein levels in the cochlea were detected by Western blot assay .Results Immunohistochemistry showed a slightly positive reaction for Rac 1 staining found in the cochlea of the control group was mainly shown in the cytoplasm and cytomembrane of ganglion and organ of Corti ;a highly positive expression was shown in GM group;and the extent of Rac 1 expression of GM+SA group was between the control and GM groups.Image analysis showed that the differences in Rac1 expression between each two groups were statistically significant ( P <0.05).Western blot assay showed that expression of Rac 1 protein was highest in GM group and decreased in GM +SA group, while the intensity of Rac1 protein expression in GM +SA group was between the control and GM group .Statistics analysis showed that the expression between each two groups had significant difference ( P <0.05 ) .Conclusions A slightly positive reaction for Rac 1 stai-ning was found in the cochlea of guinea pigs , and mainly observed in the cytoplasm and cytomembrane of spiral ganglion and organ of Corti.With the administration of gentamicin , Rac1 protein expression was upregulated in the cochlea .Simultaneous administration of antioxidant sodium salicylate could significantly decrease the expression of Rac 1 protein.These results indicated that Rac1 might play an important role in the processes of gentamicin-induced oxidative damage of cochlea .
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BACKGROUND:Tougu Xiaotong Capsule has pretty good clinical therapeutic effect on osteoarthritis of early and middle periods. However, the mechanism of Tougu Xiaotong Capsule is not ful y clarified. The RhoA GTPases can regulate chondrocyte apoptosis and hypertrophy. OBJECTIVE:To observe the Tougu Xiaotong Capsule on the expression of Rac1and Cdc42 in tumor necrosis factor-α-induced in vitro cultured rat articular chondrocytes, and to explore its mechanism of action for combating osteoarthritis. METHODS:Knee cartilage of the 4-week-old Sprague-Dawley rats was used to stably establish in vitro culture system of chondrocytes. Passage 3 chondrocytes were identified by toluidine blue staining. Chondrocyte apoptosis was successful y induced by 20μg/L tumor necrosis factor-αand then Tougu Xiaotong Capsule at different dosage (500, 100, 20 mg/L) was given after 24-hour incubation. MTT assay was used to detect cellsurvival, flow cytrometry to measure mitochondrial membrane potential, and western blot assay to determine the protein expression of Rac1, Cdc42, Bax and Bcl-2. RESULTS AND CONCLUSION:Tougu Xiaotong Capsule could reduce tumor necrosis factor-α-induced apoptosis of chondrocytes to improve the survival rate of the cells, and at the same time, could down-regulate the protein expression of Rac1, Cdc42 and Bax and increase the protein expression of Bcl-2 significantly (P<0.05). Tougu Xiaotong Capsule possibly plays a therapeutic efficacy on osteoarthritis by reducing promote apoptosis Rac1, Cdc42 and Bax expression and increasing apoptosis inhibiting gene Bcl-2 expression, thereby to inhibit apoptosis of chondrocytes.
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Triptolide, a diterpenoid triepoxide from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f., is a potential treatment for autoimmune diseases as well a possible anti-tumor agent. It inhibits proliferation of coloretal cancer cells in vitro and in vivo. In this study, its ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK1, IL6R and phosphorylated STAT3 were all reduced by triptolide treatment. Triptolide prohibited Rac1 activity and blocked cyclin D1 and CDK4 expression, leading to G1 arrest. Triptolide interrupted the IL6R-JAK/STAT pathway that is crucial for cell proliferation, survival, and inflammation. This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development.
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Animals , Humans , Male , Mice , Cell Transformation, Neoplastic/drug effects , Colitis/complications , Colonic Neoplasms/chemically induced , Dextran Sulfate/toxicity , Dimethylhydrazines/toxicity , Diterpenes/administration & dosage , Epoxy Compounds/administration & dosage , Interleukin-6/biosynthesis , Janus Kinases/metabolism , Mice, Inbred BALB C , Mice, Inbred ICR , Mice, Nude , Neoplasm Transplantation , Phenanthrenes/administration & dosage , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Tumor Burden/drug effects , rac1 GTP-Binding Protein/biosynthesisABSTRACT
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/ Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy.
Subject(s)
Animals , Mice , Phosphatidylinositol 3-Kinase/metabolism , Capsaicin/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Immunoblotting , Melanoma, Experimental/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , rac1 GTP-Binding Protein/metabolismABSTRACT
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1(Rac1-siRNA)on rat retinal neovascularization in retinae.Methods Retinal vein occlusion was indueedby retinal photodynamic medthod in 25 Sprague-Dawley rats.Rae1-siRNA vector DNA was injected intothe vitrous of one eye of those rats(gene intervention group),and empty vector DNA was injected into thefellow eye(blank control group).Rae1-siRNA vector was injected in other 25 SD rats without retinal veinocclusion(blank intervention group).Two weeks after injection,fluorescein isothiocyanate(FITC)-dextran was perfused into the hearts of all the rats,and the retinal wholemount was made to observe theneovascularization.The numbers of endothelial cells which break through the internal limiting membranewere counted after hematoxylin-eosin staining.Resuits A massive of neovascularization and FITCleakage were found in blank control group.Small part of neovaseularization and a little FITC leakage wereobserved in the gene intervention group.Retinal vessels were normal in blank intervention group.Compared with blank contrast group and blank intervention group.the difference of the mean numbers ofendothelial cells which broke through the internal limiting membrane in the gene intervention group wassignificant(F=47.168,P=0.000).Conclusion Racl-siRNA can inhibit retinal neovascularizationinduced by retinal vein occlusion in rats.