[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.

A small Rho GTPase OsRacB is required for pollen germination in rice.

Plant Rho small GTPases (Rop/Rac) are versatile molecular switches regulating many plant developmental processes. Particularly, their important functions in regulating pollen development have been demonstrated in Arabidopsis. A group of conserved Rop/Rac activators RopGEFs were recently reported to regulate rice (Oryza sativa) pollen tube germination, indicating that rice and Arabidopsis may have a conserved Rop/Rac mediated signaling pathway in regulating pollen tube growth. However, the Rop/Rac activated by the rice pollen specific RopGEFs remains to be identified. Here we demonstrated a Rop/Rac gene, OsRacB, co-expressed with the mature pollen expressed OsRopGEF2/3/6/8. The knockout mutants were normal in anther and pollen development but defective in the pollen grain germination, suggesting a specific and non-redundant role of OsRacB in the mature pollen. We further demonstrated that OsRacB is directly activated by the pollen specific expressing OsRopGEFs in vitro. Together with the previous study, we establish a RopGEF-Rop/Rac regulon which plays essential roles in rice pollen grain germination. Our data encourage further identification of the upstream and downstream players of RopGEF-Rop/Rac signaling in pollen germination and have agricultural implications for breeding robust seed yielding cultivars.

Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. AIM OF THE STUDY: To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation. MATERIALS AND METHODS: Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT + SGI-1027) and blocker group II (NLXT + Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue. RESULTS: After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P < 0.01 or P < 0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P < 0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P < 0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P < 0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P < 0.01). CONCLUSION: NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.

Shensu IV prevents glomerular podocyte injury in nephrotic rats via promoting lncRNA H19/DIRAS3-mediated autophagy.

Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography-mass spectrometry (LC-MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.

Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. AIM OF THE STUDY: In this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). MATERIALS AND METHODS: Rats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. RESULTS: Our results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 µg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. CONCLUSIONS: The present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

Manual acupuncture relieves microglia-mediated neuroinflammation in a rat model of traumatic brain injury by inhibiting the RhoA/ROCK2 pathway.

OBJECTIVE: To investigate the regulatory mechanism of manual acupuncture (MA) on microglial polarization-mediated neuroinflammation after traumatic brain injury (TBI), focusing on the RhoA/Rho-associated coiled coil-forming protein kinase (ROCK2) pathway. METHODS: Sprague Dawley (SD) rats were used to generate a TBI model using Feeney's freefall epidural impact method. MA was performed on half of the TBI model rats, while the others remained untreated. Acupuncture was administered at GV15, GV16, GV20, GV26, and LI4. At the end of the intervention, rat brain tissue samples were collected, and the microglial M1 polarization status was observed by immunofluorescence labeling of CD86, an M1 microglia-specific protein. RhoA/ROCK2 signaling components were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors. RESULTS: Compared with normal rats, the CD86 expression density in the untreated TBI model rats was high and showed an aggregated expression pattern. The genes and proteins of the RhoA/ROCK2 signaling pathway were highly expressed, and inflammatory factors were significantly increased. The CD86 expression density in TBI rats after MA was reduced compared to that in untreated TBI rats and showed a scattered distribution. The expression of RhoA/ROCK2 signaling pathway genes and proteins was also significantly reduced, and inflammatory factors were decreased. CONCLUSION: These results show that MA may inhibit M1 polarization of microglia by regulating the RhoA/ROCK2 signaling pathway, thereby reducing neuroinflammation in TBI.

Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling.

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.

Shen'ge powder decreases the cardiomyocyte hypertrophy in chronic heart failure by activating the Rho protein/Rho-associated coiledcoil forming protein kinase signaling pathway.

OBJECTIVE: The current study aimed to explore the role and the molecular mechanism of Shen'ge powder in cardiomyocyte hypertrophy in chronic heart failure (CHF). METHODS: Sprague-Dawley rats were selected for the study and divided randomly into four groups: control, model, Shen'ge powder, and fasudil group. An inverted microscope was used to determine the diameter of cardiomyocytes in each group. The survival and apoptotic rate of cardiomyocytes in each group was determined by the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The messenger RNA levels and protein expression of RhoA, Rho-associated coiled-coil forming protein kinase (ROCK), myosin light-chain phosphatase (MLCP), and myosin light-chain kinase (MLCK) were determined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. RESULTS: CHF increased the diameter and apoptotic rate of cardiomyocytes and decreased the survival rate of cardiomyocytes. The levels of RhoA, ROCK, and MLCK were increased significantly in CHF model rats, and the level of MLCP was decreased. After treating with Shen'ge powder, the expression of RhoA, ROCK, and MLCK decreased dramatically and the expression of MLCP increased. CONCLUSION: Shen'ge powder could reduce cardiomyocyte hypertrophy in CHF by regulating the Rho/ROCK signaling pathway.

Schisantherin A Improves Learning and Memory of Mice with D-Galactose-Induced Learning and Memory Impairment Through Its Antioxidation and Regulation of p19/p53/p21/Cyclin D1/CDK4/RB Gene Expressions.

Schisantherin A (SCA) was evaluated for possible function in restoring the learning and memory impairment induced by D-galactose in mice. ICR mice were treated with D-galactose subcutaneously (220 mg·kg-1), and followed by SCA in different doses (1.25, 2.50 and 5.00 mg·kg-1, administered orally) for 42 days. Effects of SCA on learning and memory were examined by step-through tests and Morris water maze tests. The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) in the peripheral blood and hippocampus of mice were assayed by water-soluble tetrazolium-1 (WST-1) and thiobarbituric acid (TBA) methods. The contents of 8 hydroxy deoxy guanosine (8-OHdG) in the hippocampus of mice were detected by immunosorbent assay methods, respectively. Quantitative real-time PCR and Western Blot were respectively used to detect the expression of p19, p53, p21, cyclin D1, CDK4 and RB genes, and the phosphorylation of RB in the hippocampus of mice. We found that SCA significantly improved the learning and memory impairment induced by D-galactose in mice. After SCA treatment, SOD activity was increased and the content of MDA was decreased in both peripheral blood and hippocampus of mice. 8-OHDG content was also decreased in the hippocampus of mice. Furthermore, the expression of p19, p53 and p21 genes was reduced and the expression of cyclin D1 and CDK4 and the phosphorylation of RB protein were elevated in the hippocampus. SCA may improve the learning and memory impairment induced by D-galactose by enhancing the antioxidant capacity, and regulating the expression of p19/p53/p21/cyclinD1/CDK4 genes, and the phosphorylation of RB protein in the hippocampus of mice.

Glycolysis regulates pollen tube polarity via Rho GTPase signaling.

As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in Arabidopsis pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a cytosolic phosphoglycerate kinase (pgkc-1) mutant with accelerated pollen germination and compromised pollen tube growth polarity. pgkc-1 mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, pgkc-1 pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the pgkc-1 phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the pgkc-1 pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.

The Rho GTPase signalling pathway in urothelial carcinoma.

Urothelial carcinoma remains a clinical challenge: non-muscle-invasive disease has a high rate of recurrence and risk of progression, and outcomes for patients with advanced disease are poor, owing to a lack of effective systemic therapies. The Rho GTPase family of enzymes was first identified >30 years ago and contains >20 members, which are divided into eight subfamilies: Cdc42, Rac, Rho, RhoUV, RhoBTB, RhoDF, RhoH, and Rnd. Rho GTPases are molecular on-off switches, which are increasingly being understood to have a critical role in a number of cellular processes, including cell migration, cell polarity, cell adhesion, cell cycle progression, and regulation of the cytoskeleton. This switch is an evolutionarily conserved system in which GTPases alternate between GDP-bound (inactive) and GTP-bound (active) forms. The activities of these Rho GTPases are many, context-dependent, and regulated by a number of proteins that are being progressively elucidated. Aberrations of the Rho GTPase signalling pathways have been implicated in various malignancies, including urothelial carcinoma, and understanding of the role of Rho GTPases in these diseases is increasing. This signalling pathway has the potential for therapeutic targeting in urothelial carcinoma. Research in this area is nascent, and much work is necessary before current laboratory-based research can be translated into the clinic.

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

The preventive effects of taurine on neural tube defects through the Wnt/PCP-Jnk-dependent pathway.

The aim of this study was to clarify the protective role of taurine in neuronal apoptosis and the role of the Wnt/PCP-Jnk pathway in mediating the preventive effects of taurine on neural tube defects (NTDs). HT-22 cells (a hippocampal neuron cell line) were divided into a control group, a glutamate-induced apoptosis group, and glutamate (4.0 mmol/L) plus low-dose taurine (L; 0.5 mmol/L) and high-dose taurine (H; 2.0 mmol/L) groups. The MTT assay was used to monitor cell proliferation and cell survival. Immunofluorescence and Western blot analyses were used to determine caspase 9 expression. Retinoic acid (RA) induced embryonic NTDs in Kunming mice, thus establishing an NTD model. Pregnant mice were divided into a control group, an RA (30 mg/kg body weight) group, and an RA (30 mg/kg body weight) plus taurine (free drinking of 2 g/L solution) group. Immunohistochemistry and Western blot analyses were used to detect the expression of Dvl, RhoA and phosphorylated (p)-Jnk/Jnk in the embryonic neural tubes. In HT-22 cells, the apoptosis rate was significantly higher and caspase 9 activation was also significantly increased in the glutamate-induced apoptosis group compared to the L and H taurine groups. In the NTD model, the expression levels of Dvl, RhoA, and p-Jnk were significantly higher in the RA group than in the control group, whereas they were significantly reduced in the RA + taurine group. This study suggests that taurine has positive effects on neuronal protection and NTD prevention. Moreover, the Wnt/PCP-Jnk-dependent pathway plays an important role in taurine-mediated prevention of NTDs.

You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui pills (YGPs) are an effective traditional Chinese formula being used clinically for the treatment of multiple sclerosis (MS). Previous studies demonstrated that YGPs exerted the potent neuroprotective effects in murine models of experimental autoimmune encephalomyelitis (EAE), which is an equivalent animal model for multiple sclerosis (MS). However, the mechanism of YGPs functions remained unclear. AIM OF THIS STUDY: The aim of this study was to evaluate the therapeutic effect of YGPs in MOG35-55-induced EAE mice and to further elucidate the underlying molecular mechanism. METHODS: Female C57BL/6 mice were divided into six groups, including the non-treated EAE model, prednisone acetate- and 1.2, 2.4 or 4.8g/kg YGPs-treated EAE groups, and a normal control group. The EAE model was established by injecting the mice subcutaneously with MOG35-55 antigen. The body weights were measured and the neurological functions were scored in each group. The pathology and morphology of the brain and spinal cord was examined. The expression of MAP-2 was detected by immunofluorescent staining. The levels of netrin1, DCC, RhoA, Rac1, and Cdc42 were assayed by immunohistochemistry, qRT-PCR and Western blot on day 40 post-immunization (PI). RESULTS: YGPs treatments significantly reduced neurological function scores in EAE mice, where the inflammatory infiltration was reduced and the axon and myelin damage in both brain and spinal cord was alleviated. In the brain and spinal cord tissues, YGPs increased the expression of neuronal factors MAP-2, netrin1 and DCC. The expression of Rac1 and Cdc42 were increased, while RhoA was reduced following YGPs treatments. CONCLUSION: Our results demonstrated that YGPs exhibited a neuroprotective effect on promoting nerve regeneration at the brain and spinal cord in EAE mice induced by MOG35-55. Netrin1, DCC and the Rho family GTPases of RhoA, Racl, Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.

Atractylodes macrocephala Koidz stimulates intestinal epithelial cell migration through a polyamine dependent mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes macrocephala Koidz (AMK), a valuable traditional Chinese herbal medicine, has been widely used in clinical practice for treating patients with disorders of the digestive system. AMK has shown noteworthy promoting effect on improving gastrointestinal function and immunity, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via intestinal epithelial (IEC-6) cell migration model. MATERIALS AND METHODS: A cell migration model of IEC-6 cells was induced by a single-edge razor blade along the diameter of the cell layers in six-well polystyrene plates. After wounding, the cells were grown in control cultures and in cultures containing spermidine (5µM, SPD, reference drug), alpha-difluoromethylornithine (2.5mM, DFMO, polyamine inhibitor), AMK (50, 100, and 200mg/L), DFMO plus SPD and DFMO plus AMK for 12h. The polyamines content was detected by high-performance liquid chromatography (HPLC) with pre-column derivatization. The Rho mRNAs expression levels were assessed by Q-RT-PCR. The Rho and non-muscle myosin II proteins expression levels were analyzed by Western blot. The formation and distribution of non-muscle myosin II stress fibers were monitored with immunostaining techniques using specific antibodies and observed by confocal microscopy. Cell migration assay was carried out using inverted microscope and the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. RESULTS: (1) Treatment with AMK caused significant increases in cellular polyamines content and Rho mRNAs and proteins expression levels, as compared to control group. Furthermore, AMK exposure increased non-muscle myosin II protein expression levels and formation of non-muscle myosin II stress fibers, and resulted in an acceleration of cell migration in IEC-6 cells. (2) Depletion of cellular polyamines by DFMO resulted in a decrease of cellular polyamines levels, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, thereby inhibiting IEC-6 cell migration. AMK not only reversed the inhibitory effects of DFMO on the polyamines content, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, but also restored cell migration to control levels. CONCLUSIONS: The results obtained from this study revealed that AMK significantly stimulates the migration of IEC-6 cells through a polyamine dependent mechanism, which could accelerate the healing of intestinal injury. These findings suggest the potential value of AMK in curing intestinal diseases characterized by injury and ineffective repair of the intestinal mucosa in clinical practice.

Protein profiling reveals antioxidant and signaling activities of NAP (Davunetide) in rodent hippocampus exposed to hypobaric hypoxia.

NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAP-mediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2D-GE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAP-supplemented hippocampus after hypoxia. Pathway analysis revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.

Revealing the mechanism of in vitro wound healing properties of Citrus tamurana extract.

In the present investigation, we examined the effect of Hyuganatsu (Citrus tamurana) extract (HE) on skin fibroblast (TIG-119) proliferation and migration during in vitro wound healing. HE selectively inhibited proliferation of TIG-119 cells at higher concentration (>1.0 mg/mL); at lower concentrations (0.1, 0.25, 0.5, and 0.75 mg/mL), it exhibited linear and time-dependent cell proliferation. In vitro scratch wound healing studies showed that the HE also accelerated the migration of cells towards the wounded region. Cytometric analysis demonstrated that HE extract did not alter G1/0 and S phases of cell cycle in any concentration studied; however, G2/M phases of cell cycle were significantly (P < 0.05) accelerated at 0.75 mg/mL dose. RT-PCR and Western blotting analysis indicated that HE markedly overexpressed levels of Rac-1, Rho-A, and Cdc-42 mRNA and the respective proteins. Cyclin-dependent kinases (Cdk-1 and -2) gene expression activity was significantly (P < 0.05) increased, but protein content decreased during treatment with HE. The induction of Cdk-1 and -2 by HE was abolished by inhibitors, transcription (DRB), and translation (CHX), implying transcriptional regulation that required de novo protein synthesis.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

2'-Hydroxyflavanone induces apoptosis through Egr-1 involving expression of Bax, p21, and NAG-1 in colon cancer cells.

SCOPE: Natural flavanones exhibit cancer preventive and/or therapeutic effects. The objective of this study was to investigate the molecular mechanism underlying the action of the antitumor activity of hydroxyflavanone using the HCT116 colon cancer cell line. METHODS AND RESULTS: We investigated the effect of hydroxyflavanones on antitumor activity. We found that 2'-hydroxyflavanone (2'-HF) potently inhibited the clonogenicity of HCT116 cells. 2'-HF triggered apoptosis in both wild-type and p53-null HCT116 cells, as revealed by DNA fragmentation and caspase activation. 2'-HF upregulated nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression through induction of Egr-1. Silencing of NAG-1 or Egr-1 using small interfering RNA (siRNA) could attenuate 2'-HF-induced apoptosis. Egr-1 also upregulated the proapoptotic gene Bax and the cell cycle inhibitor p21. CONCLUSION: Dietary 2'-HF may possess antitumor activity against human colon cancer.

1'-Acetoxychavicol acetate suppresses angiogenesis-mediated human prostate tumor growth by targeting VEGF-mediated Src-FAK-Rho GTPase-signaling pathway.

Cancer therapeutic agents that are safe, effective and affordable are urgently needed. We describe that 1'-acetoxychavicol acetate (ACA), a component of Siamese ginger (Languas galanga), can suppress prostate tumor growth by largely abrogating angiogenesis. ACA suppressed vascular endothelial growth factor (VEGF)-induced proliferation, migration, adhesion and tubulogenesis of primary cultured human umbilical vascular endothelial cells (HUVECs) in a dose-dependent manner. ACA also inhibited VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed new vasculature formation in Matrigel plugs in vivo. We further demonstrated that the mechanisms of this chavicol were to block the activation of VEGF-mediated Src kinase, focal adhesion kinase (FAK) and Rho family of small guanosine triphosphatases (GTPases) (Rac1 and Cdc42 but not RhoA) in HUVECs. Furthermore, treatment of human prostate cancer cells (PC-3) with ACA resulted in decreased cell viability and suppression of angiogenic factor production by interference with dual Src/FAK kinases. After subcutaneous administration to mice bearing human prostate cancer PC-3 xenografts, ACA (6 mg/kg/day) remarkably inhibited tumor volume and tumor weight and decreased levels of Src, CD31, VEGF and Ki-67. As indicated by immunohistochemistry and TUNEL analysis, microvessel density and cell proliferation were also dramatically suppressed in tumors from ACA-treated mice. Taken together, our findings suggest that ACA targets the Src-FAK-Rho GTPase pathway, leading to the suppression of prostate tumor angiogenesis and growth.