miR-301a Deficiency Attenuates the Macrophage Migration and Phagocytosis through YY1/CXCR4 Pathway.

(1) Background: the miR-301a is well known involving the proliferation and migration of tumor cells. However, the role of miR-301a in the migration and phagocytosis of macrophages is still unclear. (2) Methods: sciatic nerve injury, liver injury models, as well as primary macrophage cultures were prepared from the miR-301a knockout (KO) and wild type (WT) mice to assess the macrophage's migration and phagocytosis capabilities. Targetscan database analysis, Western blotting, siRNA transfection, and CXCR4 inhibition or activation were performed to reveal miR301a's potential mechanism. (3) Results: the macrophage's migration and phagocytosis were significantly attenuated by the miR-301a KO both in vivo and in vitro. MiR-301a can target Yin-Yang 1 (YY1), and miR-301a KO resulted in YY1 up-regulation and CXCR4 (YY1's down-stream molecule) down-regulation. siYY1 increased the expression of CXCR4 and enhanced migration and phagocytosis in KO macrophages. Meanwhile, a CXCR4 inhibitor or agonist could attenuate or accelerate, respectively, the macrophage migration and phagocytosis. (4) Conclusions: current findings indicated that miR-301a plays important roles in a macrophage's capabilities of migration and phagocytosis through the YY1/CXCR4 pathway. Hence, miR-301a might be a promising therapeutic candidate for inflammatory diseases by adjusting macrophage bio-functions.

Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice.

BACKGROUND: Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury. METHODS: Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the LyzCre+/-; RhoAflox/flox (cKO) mice or Lyz2Cre+/- (Cre) mice, regardless of sex. Macrophages' migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages. RESULTS: 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages. CONCLUSIONS: Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

NeuroD1 overexpression in spinal neurons accelerates axonal regeneration after sciatic nerve injury.

Neurogenic differentiation 1 (NeuroD1) is mainlyexpressed in developing neurons where it plays critical roles in neuronal maturation and neurite elongation. The potential role and mechanism of NeuroD1 in adult axonal regeneration is not clear. The present study used synapsin (SYN) Cre and AAV9-Flex vectors to conditionally overexpress NeuroD1 in adult spinal neurons and found that NeuroD1 overexpression significantly accelerated axonal regeneration and functional recovery after sciatic nerve injury. Further in vitro and in vivo experiments suggested that the mechanism of NeuroD1 promotion on axonal regeneration was related to its regulation of the expression of neurotrophin BDNF and its receptor TrkB as well as a microtubule severing protein spastin.

RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin.

RhoA-GTPase (RhoA) is widely regarded as a key molecular switch to inhibit neurite outgrowth by rigidifying the actin cytoskeleton. However, during neurite outgrowth, whether and how microtubule dynamics are regulated by RhoA remains to be elucidated. Herein, CT04 and Y27632 were used to inactivate RhoA and its downstream effector Rho-associated coiled coil-forming kinase (ROCK), while the RhoAQ63L lentiviral vector was utilized to overexpress the constitutively activated RhoA in dorsal root ganglion (DRG) neurons or neuronal differentiated PC12 cells. The current data illustrate that the RhoA signaling pathway negatively modulates neurite outgrowth and elevates the expression of Glu-tubulin (a marker for a stabilized microtubule). Meanwhile, the microtubule-severing proteins spastin and p60-katanin were downregulated by the RhoA signaling pathway. When spastin and p60-katanin were knocked down, the effects of RhoA inhibition on neurite outgrowth were significantly reversed. Taken together, this study demonstrates that the RhoA pathway-mediated inhibition of neurite outgrowth is not only related to the modulation of microfilament dynamics but is also attributable to the regulation of the expression of spastin and p60-katanin and thus influences microtubule dynamics.

Ascorbic Acid Facilitates Neural Regeneration After Sciatic Nerve Crush Injury.

Ascorbic acid (AA) is an essential micronutrient that has been safely used in the clinic for many years. The present study indicates that AA has an unexpected function in facilitating nerve regeneration. Using a mouse model of sciatic nerve crush injury, we found that AA can significantly accelerate axonal regrowth in the early stage [3 days post-injury (dpi)], a finding that was revealed by immunostaining and Western blotting for antibodies against GAP-43 and SCG10. On day 28 post-injury, histomorphometric assessments demonstrated that AA treatment increased the density, size, and remyelination of regenerated axons in the injured nerve and alleviated myoatrophy in the gastrocnemius. Moreover, the results from various behavioral tests and electrophysiological assays revealed that nerve injury-derived functional defects in motor and sensory behavior as well as in nerve conduction were significantly attenuated by treatment with AA. The potential mechanisms of AA in nerve regeneration were further explored by investigating the effects of AA on three types of cells involved in this process [neurons, Schwann cells (SCs) and macrophages] through a series of experiments. Overall, the data illustrated that AA treatment in cultured dorsal root ganglionic neurons resulted in increased neurite growth and lower expression of RhoA, which is an important inhibitory factor in neural regeneration. In SCs, proliferation, phagocytosis, and neurotrophin expression were all enhanced by AA. Meanwhile, AA treatment also improved proliferation, migration, phagocytosis, and anti-inflammatory polarization in macrophages. In conclusion, this study demonstrated that treatment with AA can promote the morphological and functional recovery of injured peripheral nerves and that this effect is potentially due to AA's bioeffects on neurons, SCs and macrophages, three of most important types of cells involved in nerve injury and regeneration.

Inhibition of RhoA-Subfamily GTPases Suppresses Schwann Cell Proliferation Through Regulating AKT Pathway Rather Than ROCK Pathway.

Inhibiting RhoA-subfamily GTPases by C3 transferase is widely recognized as a prospective strategy to enhance axonal regeneration. When C3 transferase is administered for treating the injured peripheral nerves, Schwann cells (SCs, important glial cells in peripheral nerve) are inevitably impacted and therefore SC bioeffects on nerve regeneration might be influenced. However, the potential role of C3 transferase on SCs remains elusive. Assessed by cell counting, EdU and water-soluble tetrazolium salt-1 (WST-1) assays as well as western blotting with PCNA antibody, herein we first found that CT04 (a cell permeable C3 transferase) treatment could significantly suppress SC proliferation. Unexpectedly, using Y27632 to inhibit ROCK (the well-accepted downstream signal molecule of RhoA subfamily) did not impact SC proliferation. Further studies indicated that CT04 could inactivate AKT pathway by altering the expression levels of phosphorylated AKT (p-AKT), PI3K and PTEN, while activating AKT pathway by IGF-1 or SC79 could reverse the inhibitory effect of CT04 on SC proliferation. Based on present data, we concluded that inhibition of RhoA-subfamily GTPases could suppress SC proliferation, and this effect is independent of conventional ROCK pathway but involves inactivation of AKT pathway.

CHP2 Promotes Cell Proliferation in Breast Cancer via Suppression of FOXO3a.

Calcineurin B homologous protein isoform 2 (CHP2), an essential cofactor for Na+/H+ exchanger isoform 1 (NHE1), is identified to be expressed in various malignant cell lines. However, the clinical significance and biological role of CHP2 in breast cancer remain to be established. Here, CHP2 was markedly overexpressed in breast cancer cells and clinical tumor specimens. Immunohistochemical analysis revealed that the expression of CHP2 was significantly correlated with patients' clinicopathologic characteristics like clinical stage, and breast cancer patients with high CHP2 expression had shorter overall survival compared with patients with low CHP2 expression. Moreover, it was demonstrated that overexpressing CHP2 significantly enhanced, whereas silencing endogenous CHP2 inhibited, the proliferation and tumorigenicity of breast cancer cells in vitro and in vivo In addition, overexpression of CHP2 accelerated, whereas inhibition of CHP2 retarded, G1-S phase cell-cycle transition in breast cancer cells. Mechanistically, overexpression of CHP2 activated AKT signaling and suppressed the transactivation of the forkhead box O3 (FOXO3/FOXO3a) transcription factor.Implications: This study discovers a previously unrecognized role of CHP2 in the progression of breast cancer and supports the significance of this gene as a novel prognostic biomarker and a potential therapeutic target for breast cancer. Mol Cancer Res; 16(10); 1512-22. ©2018 AACR.

[Survey on thalassemia among people of reproductive age in Guilin City, Guangxi, China].

OBJECTIVE: To study the present situation of thalassemia among people at reproductive age in Guilin city. METHODS: A complete red blood cell mean cell volume (RBCMCV) was detected as well as hemoglobin electrophoresis analysis were done for all samples. Suspected alpha or beta thalassemia cases, were screened out and alpha and beta thalassemia genes were detected by PCR-RDB. RESULTS: Among 1580 cases, 79 cases were detected a thalassemia gene positive, with the detection rate as 5.00%. The detection rates on a thalassemia were 5.32%, 4.68% in males and females, but no significant difference (chi2 = 3.04, chi2 < chi0.05 (1) = 3.84, P > 0.05). 114 cases were detected carrying beta thalassemia gene with the detection rate as 7.22%. The detection rates on beta thalassemia were 7.85%, 6.58% in males and females, with no significant difference (chi2 = 0.95, chi2 < chi0.05(l) = 3.84, P > 0.05). The overall detection rate of thalassemia was 12.22% (193/1580). Alpha thalassemia were found to have had ten genotypes, with --SEA/alphaalpha the most common one and the detection rate was 3.54%. -Alpha(3.7)/alphaalpha appeared the majority in the static alpha thalassemia and --SEA/alphaalpha took the majority in the light a thalassemia, while --SEA-alphaCS was the major one in the intermedia alpha thalassemia, with gene contributions of 7.59%, 70.88% and 2.53%. Beta thalassemia was detected having seven genotypes,with CD41-42 (-TTCT) the most common one in beta thalassemia, and the detection rate was 3.16%. The commonly seen three mutations, CD41-42 (-TTCT), CD17 (A-->T) and IVS-II-654 (C-->T) were accounted for 87.71% of beta thalassemia. The detection rate on thalassemia a and beta combination was 0.63%. CONCLUSION: The detection rate of thalassemia among people at productive age in Guilin city was relatively high.

[Immunonanogold catalytic spectrophotometric determination of trace IgG].

Gold nanoparticles the size of about 10 nm were prepared by improved trisodium citrate reduction procedure, and were used to label goat anti-human IgG to obtain a sensitive spectral probe for IgG in the condition of pH 6.5. The immune reaction of nanogold-labeled IgG antibody (anti-IgG) with the antigen IgG took place to form the nanogold immune complex in pH 7.O Na2HPO4-C6H8O7 buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 7.0, 10.76 microg x mL(-1) nanogold-labeled anti-IgG, 8.0% PEG 6000 and incubation time of 30 min under the ultrasonic irradiation. After centrifuging for 15 min at 16000 rpm, the excess nanogold-labeled anti-IgG in the upper solutions was obtained, and was used to catalyze the colored particle reaction of HAuCl4 with NH2 OH x HCl to produce gold particles with bigger size. The influence of pH value, HAuCl4 and NH2OH x HCl concentration, and reaction temperature and time on the immunonanogold catalytic reaction was considered spectrophotometrically. A pH 2.27 Na3C6H5O7-HCl buffer solution, 0.094 mmol x L HAuCl4, 1.92 mmol x L NH22OH x HCl, and reaction time of 6 min at 30 degrees C water bath were chosen for use. Results demonstrated that with increasing IgG, the concentration of gold labeled anti-IgG in the upper solution decreased, and the absorbance decreased linearly. Linear relationships between the decreased absorbance at 700 nm and the IgG concentration CIgG in the range of 0.10-10 ng x mL(-1) were obtained. Its regress equation was deltaA(760 nm) = 0.0144c(IgG) + 0.0042, the related coefficient was 0.9926, and the detection limit reached 0.06 ng x mL(-1) IgG. The influence of foreign substances on the determination of 3 ng x mL(-1) IgG was examined, with the relative error +/-10%. Results showed that the following coexistent substances had no impact on the assay: 6000 ng x mL(-1) HSA, 6000 ng x mL(-1) gluocose, 6000 ng x ml(-1) Zn(II), 3000 ng x mL(-1) IgA, 3000 ng x mL(-1) Ca(II), 3000 ng x mL(-1) L-arginine, 3000 ng x mL(-1) beta-phenylalanine, 2400 ng x mL(-1) Cu(II), 2400 ng x mL(-1) EDTA, 2400 ng x mL(-1) L-cystinol etc. This showed that the assay has high selectivity. The sensitive, rapid and highly specific assay was applied to the quantification of IgG in human sera, with satisfactory results.

[Immune resonance scattering spectral method for the determination of trace IgG].

In pH 7.0 Tris-HCl buffer solution, goat-anti-rabbit IgG is combined with rabbit IgG specifically, and aggregates to form immune complex particles that exhibit three resonance scattering peaks at 330, 400 and 520 nm respectively, and a synchronous scattering peak at 470 nm, in the presence of PEG-20000. The scattering intensity at 470 nm is linear to the rabbit IgG concentration in the range of 1.33 to 133.3 microg x mL(-1). The detection limit is 0.99 microg x mL(-1). The method was applied to the quantitative analysis of rabbit IgG, with satisfactory results.