Exploration of the Mechanisms Underlying Yu's Enema Formula in Treating Ulcerative Colitis by Blocking the RhoA/ROCK Pathway based on Network Pharmacology, High-performance Liquid Chromatography Analysis, and Experimental Verification.

BACKGROUND: The traditional Chinese medicine formula, Yu's Enema Formula (YEF), has demonstrated potential in the treatment of Ulcerative Colitis (UC). OBJECTIVE: This study aimed to unveil the anti-UC mechanisms of YEF. METHODS: Utilizing public databases, we obtained YEF and UC-related targets. GO and KEGG analyses were conducted via clusterProfiler and Reactome. The STRING database facilitated the construction of the PPI network, and hub targets were selected using cytoHubba. We used R software for differential expression and correlation analyses, and molecular docking was performed with PyMOL and AutoDock. HPLC analysis identified the compounds in YEF. For in vivo validation, a UC rat model was employed. RESULTS AND DISCUSSION: 495 YEF-UC overlapping targets were identified. GO and KEGG analyses indicated enrichment in exogenous stimuli response, peptide response, positive MAPK cascade regulation, interleukin- related signaling, and the TLR4 cascade. Hub targets included CTNNB1, JUN, MAPK1, MAPK3, SRC, STAT3, TLR4, TP53, and RELA, which were often interconnected. Molecular docking revealed quercetin's strong binding affinity with CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, consistent with HPLC analysis. In vivo experiments suggested that YEF has the potential to alleviate UC symptoms and protect the intestinal mucosal barrier by inhibiting the RhoA/ROCK pathway. CONCLUSION: YEF may safeguard the intestinal mucosal barrier in UC by targeting CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, while blocking the RhoA/ROCK pathway.

[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.

The loop-tail mouse model displays open and closed caudal neural tube defects.

Neural tube defects (NTDs) are the second most common cause of congenital malformations and are often studied in animal models. Loop-tail (Lp) mice carry a mutation in the Vangl2 gene, a member of the Wnt-planar cell polarity pathway. In Vangl2+/Lp embryos, the mutation induces a failure in the completion of caudal neural tube closure, but only a small percentage of embryos develop open spina bifida. Here, we show that the majority of Vangl2+/Lp embryos developed caudal closed NTDs and presented cellular aggregates that may facilitate the sealing of these defects. The cellular aggregates expressed neural crest cell markers and, using these as a readout, we describe a systematic method to assess the severity of the neural tube dorsal fusion failure. We observed that this defect worsened in combination with other NTD mutants, Daam1 and Grhl3. Besides, we found that in Vangl2+/Lp embryos, these NTDs were resistant to maternal folic acid and inositol supplementation. Loop-tail mice provide a useful model for research on the molecular interactions involved in the development of open and closed NTDs and for the design of prevention strategies for these diseases.

MiR-936 Targets GPR78 and Regulates Chemotherapy Resistance in Non-Small Cell Lung Cancer by Activating the Galphaq Rho GTPase Pathway.

Objective: We aimed to explore the mechanism of microRNA-936 (miR-936) targeting G protein coupled receptor 78 (GPR78) regulating chemoresistance of non-small cell lung cancer (NSCLC) by activating the Galphaq Rho GTPase pathway. Methods: We added cisplatin to DMEM medium of HCC827/cisplatin cells and adjusted the final concentration to 1 µg/mL. Cells were divided into the control group and the miR-936 transfection group. Tissue samples were divided into the normal tissue group and the NSCLC tissue group. The mRNA expression of miR-936 in tissue samples was analyzed via reverse transcription polymerase chain reaction (RT-PCR). Cell migration and invasion were detected by wound healing assay. Cell counting kit 8 (CCK-8) was used to detect the cell viability 1, 2 and 3 days after cisplatin induction. The toxicity of cisplatin was analyzed by flow cytometry. The targeting relationship between miR-936 and GPR78 was detected by luciferase reporter gene assay. The regulation of miR-936 on GPR78/Rho GTPase was analyzed by Western blot. Results: The expression of miR-936 in NSCLC was lower than in normal tissues (P < .05). The number of cell migrations and invasions in the miR-936 transfection group was lower than in the control group (P < .05). The cell viability in the miR-936 transfection group was lower than in the control group on the 1st, 2nd and 3rd day (P < .05). With the increase in cisplatin concentration, the apoptosis rate of cells increased in a dependent manner (P < .05). Compared with GPR78 Mut, overexpression of miR-936 inhibited the luciferase activity of GPR78 WT 3'- UTR (P < .05). The expression of GPR78, RhoA, Rac1 and ABCB1 protein in the miR-936 transfection group was lower than in the control group (P < .05). The expression of GPR78 protein in the inhibitor+miR-936 transfection group was lower than in the inhibitor+control group (P < .05). Conclusion: miR-936 targets GPR78 and improves the sensitivity of NSCLC cells to cisplatin via the Galphaq Rho GTPase pathway.

Alzheimer's disease large-scale gene expression portrait identifies exercise as the top theoretical treatment.

Alzheimer's disease (AD) is a complex neurodegenerative disorder that affects multiple brain regions and is difficult to treat. In this study we used 22 AD large-scale gene expression datasets to identify a consistent underlying portrait of AD gene expression across multiple brain regions. Then we used the portrait as a platform for identifying treatments that could reverse AD dysregulated expression patterns. Enrichment of dysregulated AD genes included multiple processes, ranging from cell adhesion to CNS development. The three most dysregulated genes in the AD portrait were the inositol trisphosphate kinase, ITPKB (upregulated), the astrocyte specific intermediate filament protein, GFAP (upregulated), and the rho GTPase, RHOQ (upregulated). 41 of the top AD dysregulated genes were also identified in a recent human AD GWAS study, including PNOC, C4B, and BCL11A. 42 transcription factors were identified that were both dysregulated in AD and that in turn affect expression of other AD dysregulated genes. Male and female AD portraits were highly congruent. Out of over 250 treatments, three datasets for exercise or activity were identified as the top three theoretical treatments for AD via reversal of large-scale gene expression patterns. Exercise reversed expression patterns of hundreds of AD genes across multiple categories, including cytoskeleton, blood vessel development, mitochondrion, and interferon-stimulated related genes. Exercise also ranked as the best treatment across a majority of individual region-specific AD datasets and meta-analysis AD datasets. Fluoxetine also scored well and a theoretical combination of fluoxetine and exercise reversed 549 AD genes. Other positive treatments included curcumin. Comparisons of the AD portrait to a recent depression portrait revealed a high congruence of downregulated genes in both. Together, the AD portrait provides a new platform for understanding AD and identifying potential treatments for AD.

Liquiritigenin alleviates doxorubicin-induced chronic heart failure via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway.

Chronic heart failure (CHF) is one of the most common chronic diseases with increasing incidence and mortality. Liquiritigenin (LQG) is shown to protect mice from cardiotoxicity. However, its underlying mechanism remains unclear. Our study aimed to reveal the role of ARHGAP18 in LQG-mediated cardioprotective effects in CHF. In the current study, CHF cell model and rat model were established by the application of doxorubicin (DOX). The reactive oxygen species (ROS) level and cell apoptosis were determined by flow cytometry. The cardiac function of rats was evaluated by measuring left ventricular systolic pressure, left ventricular end diastolic pressure, and serum level of lactate dehydrogenase and brain natriuretic peptide. The expression of active RhoA was elevated and that of ARHGAP18 was decreased in DOX-induced CHF cell model. ARHGAP18 could reduce DOX-induced RhoA activation, ROS elevation, and cell apoptosis. Meanwhile, the knockdown of ARHGAP18 could promote the activation of RhoA, the level of ROS, and the rate of cell apoptosis, which could be reversed by the application of RhoA inhibitor. LQG promoted the expression of ARHGAP18 and exerted similar effects of ARHGAP18 in CHF cell model. The application of LQG could also reverse the effects mediated by ARHGAP18 knockdown. Moreover, LQG significantly improved cardiac function and ameliorated DOX-induced cardiotoxicity of CHF rats. In conclusion, LQG could alleviate DOX-induced CHF via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway. LQG was a potential agent for CHF treatment.

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.

Restitution of epithelial cells during intestinal mucosal wound healing: The effect of a polysaccharide from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerotis (Cooke) Ryvarden cultivar TM02, also known as tiger's milk mushroom, is regarded as important folk medicine in Malaysia, while is used for the treatment of liver cancer, chronic hepatitis, gastric ulcer in traditional Chinese medicine. However, there is no compilation of scientific evidence that its protection for gastric, and no attempts have been made to understand how polysaccharides in Lignosus rhinocerotis might promote intestinal mucosal wound healing. AIM OF THE STUDY: This study aimed to investigate the effect and mechanism of ß-glucan prepared from L. rhinocerotis using an enzymatic method on epithelial restitution during intestinal mucosal damage. MATERIALS AND METHODS: Based on FT-IR, MALDI-TOF-MS, HPSEC-MALLS-RID, and AFM, the structure of polysaccharides from L. rhinocerotis was analysed. In addition, polysaccharides were used to test for wound healing activity in IEC-6 cells by measuring cell migration, proliferation, and expression of cell division control protein 42, Rac-1, RhoA, and Par-3. RESULTS: ß-glucan was extracted using enzyme-assisted extraction, and a yield of approximately 8.5 ± 0.8% was obtained from the dried biomass. The ß-glucan extracted by enzyme-assisted extraction (EAE) of polysaccharides was composed entirely of D-glucose with a total carbohydrate content of 95.5 ± 3.2%. The results of HPLC, FTIR, and MALDI-TOF-MS analyses revealed EAEP to be confirmed as ß-glucan. The molecular weight of prepared ß-glucan was found to be 5.315 × 104 g/mol by HPSEC-MALLS-RID. Furthermore, mucosal wound healing studies showed that the treatment of IEC-6 with a ß-glucan concentration of 200 µg/mL promoted cell migration and proliferation, and it enhanced the protein expression of cell division control protein 42, Rac-1, RhoA, and Par-3. CONCLUSIONS: The present study reveals that the prepared ß-glucan accelerates intestinal epithelial cell proliferation and migration via activation of Rho-dependent pathway. Hence, ß-glucan can be employed as a prospective therapeutic agent for the treatment of diseases associated with gastrointestinal mucosal damage, such as peptic ulcers and inflammatory bowel disease.

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.

The mechanism of electroacupuncture for treating spinal cord injury rats by mediating Rho/Rho-associated kinase signaling pathway.

Objective: To determine the changes of gene and protein expression through Rho/ROCK signaling pathway in EA treated spinal cord injury (SCI) rats and to unveil the possible underlying mechanism.Design: Animal study.Setting: Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine.Participants: Eighty Male Sprague Dawley rats.Interventions: Electroacupuncture at Yaoyangguan (GV3), Dazhui (GV14), Zusanli (ST36) and Ciliao (BL32) and/or blocking agent Y27632 treatment.Outcome Measures: Protein expression was detected by ELISA and Western blotting, mRNA expression was detected by quantitative PCR and in situ hybridization. Morphological changes in spinal cord were evaluated by HE-staining and Nissl staining. Hindlimb motor function in the rats was evaluated by Basso-Beattie-Bresnahan (BBB) assessment methods.Results: Compared with injured rats in SCI group, EA, blocking agent Y27632 and EA + blocking agent Y27632 treatment had significantly reduced mRNA and protein expression levels of RhoA and ROCKII, decreased p-MLC protein expression and p-MLC/MLC ratio, suppressed cPLA2 activity and PGE2 level, improved spinal cord tissue morphology and BBB score of lower limb movement function at 7 days and at 14 days (P < 0.01 or <0.05).Conclusion: Similar to the blocking agent Y27632, EA may have a notable inhibitory effect on the Rho/ROCK signaling pathway after SCI, therefore reducing the inhibition of axonal growth and inflammatory reaction may be a key mechanism of EA treatment for SCI.

Rho-GTPase pathways may differentiate treatment response to TNF-alpha and IL-17A inhibitors in psoriatic arthritis.

Biological therapies have dramatically improved the therapeutic landscape of psoriatic arthritis (PsA); however, 40-50% of patients are primary non-responders with response rates declining significantly with each successive biological therapy. Therefore, there is a pressing need to develop a coherent strategy for effective initial and subsequent selection of biologic agents. We interrogated 40 PsA patients initiating either tumour necrosis factor inhibitors (TNFi) or interleukin-17A inhibitors (17Ai) for active PsA. Patients achieving low disease activity according to the Disease Activity Index for PsA (DAPSA) at 3 months were classified as responders. Baseline and 3-month CD4+ transcript profiling were performed, and novel signaling pathways were identified using a multi-omics profiling and integrative computational analysis approach. Using transcriptomic data at initiation of therapy, we identified over 100 differentially expressed genes (DEGs) that differentiated IL-17Ai response from non-response and TNFi response from non-response. Integration of cell-type-specific DEGs with protein-protein interactions and further comprehensive pathway enrichment analysis revealed several pathways. Rho GTPase signaling pathway exhibited a strong signal specific to IL-17Ai response and the genes, RAC1 and ROCKs, are supported by results from prior research. Our detailed network and pathway analyses have identified the rewiring of Rho GTPase pathways as potential markers of response to IL17Ai but not TNFi. These results need further verification.

Nelumbo nucifera leaf polyphenol extract and gallic acid inhibit TNF-α-induced vascular smooth muscle cell proliferation and migration involving the regulation of miR-21, miR-143 and miR-145.

Nelumbo nucifera leaf water extract (NLE) attenuates high-fat diet (HFD)-induced rabbit atherosclerosis, but its mechanism of action and the relevant compounds remain unclear. Modulating the proliferation and migration of vascular smooth muscle cells (VSMCs) may be an enforceable strategy for atherosclerosis prevention. Therefore, we investigated the potential mechanisms of N. nucifera leaf polyphenol extract (NLPE) and its active ingredient gallic acid (GA) in VSMC proliferation and migration. A7r5 rat aortic VSMCs were provoked using 50 ng mL-1 tumor necrosis factor (TNF)-α; the NLPE or GA reduced the TNF-α-induced migration by inhibiting the transforming protein RhoA/cell division cycle protein 42 pathway. The NLPE or GA suppressed the TNF-α-induced VSMC proliferation by inhibiting the Ras pathway and increasing the expression of phosphatase and tensin homolog (PTEN), kinase suppressor of Ras 2, and inducible nitric oxide synthase. The NLPE or GA increased PTEN expression by downregulating microRNA (miR)-21 expression and reduced Ras and RhoA expression by upregulating miR-143 and miR-145 expression. The NLPE and GA use potentially prevents atherosclerosis by inhibiting the VSMC migration and proliferation. The mechanisms involve the regulation of the miRNA in PTEN, the Ras/extracellular-signal-regulated kinase pathway, and Rho family proteins.

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.

Exocytosis and endocytosis: coordinating and fine-tuning the polar tip growth domain in pollen tubes.

Pollen tubes rapidly elongate, penetrate, and navigate through multiple female tissues to reach ovules for sperm delivery by utilizing a specialized form of polar growth known as tip growth. This process requires a battery of cellular activities differentially occurring at the apical growing region of the plasma membrane (PM), such as the differential cellular signaling involving calcium (Ca2+), phospholipids, and ROP-type Rho GTPases, fluctuation of ions and pH, exocytosis and endocytosis, and cell wall construction and remodeling. There is an emerging understanding of how at least some of these activities are coordinated and/or interconnected. The apical active ROP modulates exocytosis to the cell apex for PM and cell wall expansion differentially occurring at the tip. The differentiation of the cell wall involves at least the preferential distribution of deformable pectin polymers to the apex and non-deformable pectin polymers to the shank of pollen tubes, facilitating the apical cell expansion driven by high internal turgor pressure. Recent studies have generated inroads into how the ROP GTPase-based intracellular signaling is coordinated spatiotemporally with the external wall mechanics to maintain the tubular cell shape and how the apical cell wall mechanics are regulated to allow rapid tip growth while maintaining the cell wall integrity under the turgor pressure. Evidence suggests that exocytosis and endocytosis play crucial but distinct roles in this spatiotemporal coordination. In this review, we summarize recent advances in the regulation and coordination of the differential pectin distribution and the apical domain of active ROP by exocytosis and endocytosis in pollen tubes.

Sodium aescinate significantly suppress postoperative peritoneal adhesion by inhibiting the RhoA/ROCK signaling pathway.

BACKGROUND: Although mechanical barriers and modern surgical techniques have been developed to prevent postoperative adhesion formation, high incidence of adhesions still represents an important challenge in abdominal surgery. So far, there has been no available therapeutic drug in clinical practice. PURPOSE: In this study, we explored the efficacy of sodium aescinate (AESS) treatment against postoperative peritoneal adhesions, the potential molecular mechanism was also investigated. STUDY DESIGN AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 6 groups for the study: the blank, vehicle, positive control and three AESS administration groups (0.5, 1 and 2 mg/kg/d, intravenous administration for 7 days). Adhesions were induced by discretely ligating peritoneal sidewall. An IL-1ß-induced HMrSV5 cell model was also performed to explore possible functional mechanism. RESULTS: The results indicated that the incidence and severity of peritoneal adhesions were significantly lower in the AESS-treated groups than that in the vehicle and positive control group. AESS-treated groups showed that the secretion, activity, and expression of tPA in rat peritoneum were notably increased. The FIB levels in rat plasma were decreased. The immunohistochemical staining analysis demonstrated that collagen I and α-SMA deposition were significantly attenuated in AESS-treated peritoneal tissues. Besides, we found that AESS treatment reduced the protein levels of p-MYPT1. To further explore the mechanisms of AESS, both activator and inhibitors of RhoA/ROCK pathway were employed in this study. It was found that AESS-induced up-regulation of tPA was reversed by activator of ROCK, but the effects of ROCK inhibitors were consistent with AESS. CONCLUSION: Taken together, the findings of in vivo and in vitro experiments proved that AESS could significantly suppress postoperative peritoneal adhesion formation through inhibiting the RhoA/ROCK signaling pathway. Our researches provide important pharmacological basis for AESS development as a potential therapeutic agent on peritoneal adhesions.

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.

Physiologic Electrical Fields Direct Retinal Ganglion Cell Axon Growth In Vitro.

Purpose: The purpose of this study was to characterize the ability of applied electrical fields (EFs) to direct retinal ganglion cell (RGC) axon growth as well as to assess whether Rho GTPases play a role in translating electrical cues to directional cues. Methods: Full-thickness, early postnatal mouse retina was cultured in electrotaxis chambers and exposed to EFs of varying strengths (50-200 mV/mm). The direction of RGC axon growth was quantified from time-lapsed videos. The rate of axon growth and responsiveness to changes in EF polarity were also assessed. The effect of toxin B, a broad-spectrum inhibitor of Rho GTPase signaling, and Z62954982, a selective inhibitor of Rac1, on EF-directed growth was determined. Results: In the absence of an EF, RGC axons demonstrated indiscriminate directional growth from the explant edge. Retinal cultures exposed to an EF of 100 and 200 mV/mm showed markedly asymmetric growth, with 74.2% and 81.2% of axons oriented toward the cathode, respectively (P < 0.001). RGC axons responded to acute changes in EF polarity by redirecting their growth toward the "new" cathode. This galvanotropic effect was partially neutralized by toxin B and Rac1 inhibitor Z62954982. Conclusions: RGC axons exhibit cathode-directed growth in the presence of an EF. This effect is mediated in part by the Rho GTPase signaling cascade.

Preserving Mitochondrial Structure and Motility Promotes Recovery of White Matter After Ischemia.

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.