Phytochrome interacting factor 3 mediates low light signaling to regulate isorhynchophylline biosynthesis in Uncaria rhynchophylla.

Phytochrome interacting factors (PIFs) serve as crucial regulators in the light signal transduction pathway and also mediate light signals to regulate secondary metabolite synthesis in plants. However, the regulator role of PIFs in secondary metabolites often varies among different plants. Isorhynchophylline (IRN), an iconic secondary metabolite of Uncaria rhynchophylla, holds significant medicinal value. Low light induces the synthesis of IRN in previous research, but PIFs in U. rhynchophylla have not been studied to date. Building on this, we identified a PIF protein, UrPIF3, which possesses the typical conserved domains of the PIFs and is localized in the nucleus. Moreover, the expression level of UrPIF3 is consistently positively correlated with the expression of two key enzyme genes (UrSGD and UrSTR) in the IRN biosynthesis pathway, regardless of whether under low light or restoring light conditions. Yeast one-hybrid and dual-luciferase assays further demonstrated that UrPIF3 can directly upregulate UrSGD. Conversely, silencing UrPIF3 inhibits IRN synthesis, and significantly reduces the expression levels of UrSGD and UrSTR. In summary, our results suggest that under low light conditions, UrPIF3 can directly upregulate UrSGD and indirectly upregulate UrSTR, thereby promoting the synthesis of IRN.

Delayed simvastatin treatment improves neurological recovery after cryogenic traumatic brain injury through downregulation of ELOVL1 by inhibiting mTOR signaling.

Statins are well-tolerated and widely available lipid-lowering medications with neuroprotective effects against traumatic brain injury (TBI). However, whether delayed statin therapy starting in the subacute phase promotes recovery after TBI is unknown. Elongation of the very long-chain fatty acid protein 1 (ELOVL1) is involved in astrocyte-mediated neurotoxicity, but its role in TBI and the relationship between ELOVL1 and statins are unclear. We hypothesized that delayed simvastatin treatment promotes neurological functional recovery after TBI by regulating the ELOVL1-mediated production of very long-chain fatty acids (VLCFAs). ICR male mice received daily intragastric administration of 1, 2 or 5 mg/kg simvastatin on Days 1-14, 3-14, 5-14, or 7-14 after cryogenic TBI (cTBI). The results showed that simvastatin promoted motor functional recovery in a dose-dependent manner, with a wide therapeutic window of at least 7 days postinjury. Meanwhile, simvastatin inhibited astrocyte and microglial overactivation and glial scar formation, and increased total dendritic length, neuronal complexity and spine density on day 14 after cTBI. The up-regulation of ELOVL1 expression and saturated VLCFAs concentrations in the cortex surrounding the lesion caused by cTBI was inhibited by simvastatin, which was related to the inhibition of the mTOR signaling. Overexpression of ELOVL1 in astrocytes surrounding the lesion using HBAAV2/9-GFAP-m-ELOVL1-3xFlag-EGFP partially attenuated the benefits of simvastatin. These results showed that delayed simvastatin treatment promoted functional recovery and brain tissue repair after TBI through the downregulation of ELOVL1 expression by inhibiting mTOR signaling. Astrocytic ELOVL1 may be a potential target for rehabilitation after TBI.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

OBJECTIVE: In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). RESULTS: The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20 g·l-1 cellulase and 15 g·l-1 macerozyme for 6 h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. CONCLUSIONS: We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Delayed Chronic Acidic Postconditioning Improves Poststroke Motor Functional Recovery and Brain Tissue Repair by Activating Proton-Sensing TDAG8.

Acidic postconditioning by transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO2 for various durations (three cycles of 10- or 20-min CO2 inhalation/10-min break) at days 3-7, 7-21, or 3-21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO2 concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3-7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.

The study on interacting factors and functions of GASA6 in Jatropha curcas L.

BACKGROUND: The gibberellic acid-stimulated Arabidopsis (GASA) gene encodes a class of cysteine-rich functional proteins and is ubiquitous in plants. Most GASA proteins are influence the signal transmission of plant hormones and regulate plant growth and development, however, their function in Jatropha curcas is still unknown. RESULTS: In this study, we cloned JcGASA6, a member of the GASA family, from J. curcas. The JcGASA6 protein has a GASA-conserved domain and is located in the tonoplast. The three-dimensional structure of the JcGASA6 protein is highly consistent with the antibacterial protein Snakin-1. Additionally, the results of the yeast one-hybrid (Y1H) assay showed that JcGASA6 was activated by JcERF1, JcPYL9, and JcFLX. The results of the Y2H assay showed that both JcCNR8 and JcSIZ1 could interact with JcGASA6 in the nucleus. The expression of JcGASA6 increased continuously during male flower development, and the overexpression of JcGASA6 was associated with filament elongation of the stamens in tobacco. CONCLUSION: JcGASA6, a member of the GASA family in J. curcas, play an important role in growth regulation and floral development (especially in male flower). It is also involved in the signal transduction of hormones, such as ABA, ET, GA, BR, and SA. Also, JcGASA6 is a potential antimicrobial protein determined by its three-dimensional structure.

Nab-paclitaxel plus S-1 versus oxaliplatin plus S-1 as first-line treatment in advanced gastric cancer: results of a multicenter, randomized, phase III trial (GAPSO study).

Background: This study aimed to investigate the superiority of nab-paclitaxel plus S-1 (AS) over oxaliplatin plus S-1 (SOX) in patients with advanced gastric cancer (AGC). Methods: In this multicenter, randomized, phase III superiority trial, eligible patients with unresectable, locally advanced gastric adenocarcinoma were recruited and randomly assigned (1:1) to receive AS (nab-paclitaxel 260 mg/m2 on day 1 or 130 mg/m2 on days 1 and 8; oral S-1 40-60 mg twice daily for 14 days) or SOX (130 mg/m2 oxaliplatin on day 1; oral S-1 40-60 mg twice daily for 14 days) every 3 weeks for up to six cycles. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival, objective response rate, and safety. Results: Owing to slow enrolment, an unplanned interim analysis was performed, resulting in the early termination of the study on 31 December 2021 (data cutoff). Between March 2019 and March 2021, 97 patients (AS, n = 48; SOX, n = 49) were treated and evaluated for efficacy and safety of AS and SOX. As of the data cutoff, the median follow-up was 23.13 months [95% confidence interval (CI), 13.39-32.87]. The median PFS was 9.03 months (95% CI, 6.50-11.56) in the AS group and 5.07 months (95% CI, 4.33-5.81) in the SOX group, demonstrating a better PFS tendency following AS treatment than SOX treatment (hazard ratio = 0.59; 95% CI, 0.37-0.94; p = 0.03). The most common grade 3 or worse adverse events were anemia, neutropenia, and leukopenia in both groups, with a higher incidence of thrombocytopenia in the SOX group. Conclusion: Although this study was terminated early, the results demonstrated a better PFS tendency in patients with AGC who were treated with AS than in those treated with SOX, with controllable toxicities. Trial registration: Clinical Trials.gov identifiers: NCT03801668. Registered January 11, 2019.

Comparative transcriptome analysis revealed the molecular mechanism of the effect of light intensity on the accumulation of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN), the main medicinal components in plant Uncaria rhynchophylla, have potential effects on Alzheimer's disease. Understanding the influence of environmental factors, especially light intensity, on the production of these active ingredients will help to improve cultivation techniques. Compared with the 100% light intensity (CK), the contents of RIN and IRN in U. rhynchophylla leaves significantly increased at 20% light intensity (HS) after 7 and 21 days. Short-term shading (21d) changed some morphological indicators of U. rhynchophylla, but did not affect its biomass. Transcriptome profile analysis was performed on data from two groups (7 and 21 days) of CK and HS samples and yielded 79,817 unigenes with an average length of 1023 bp. Concurrently, 2391 and 2136 differentially expressed genes were identified in the transcriptome data for, respectively, 7 and 21 days of shade treatment. Notably, unigenes known to be involved upstream in the biosynthesis of RIN and IRN, such as G8O, IO, 7-DLGT, LAMT, TDC, and STR, were mostly upregulated. In addition, 1065 putative transcription factors (TFs) were identified and grouped into 55 TF families; 26 TFs showed differential expression in the shade treatment after 7 and 21 days. HY5 and PIFs, two important TFs of the light signaling pathway, also showed differential expression. This study provides insight into how gene expression was affected by light intensity during RIN and IRN accumulation in U. rhynchophylla. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01142-2.

Transcriptome revealing the dual regulatory mechanism of ethylene on the rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN) are extracted from Uncaria rhynchophylla, which are used to treat Alzheimer's disease. However, the massive accumulation of RIN and IRN in U. rhynchophylla requires exogenous stimulation. Ethylene is a potential stimulant for RIN and IRN biosynthesis, but there is no study on the role of ethylene in RIN or IRN synthesis. This study investigated the regulation of ethylene in RIN and IRN biosynthesis in U. rhynchophylla. An increase in the content of RIN and IRN was observed that could be attributed to the release of ethylene from 18 mM ethephon, while ethylene released from 36 mM ethephon reduced the content of RIN and IRN. The transcriptome and weighted gene co-expression network analysis indicated the up-regulation of seven key enzyme genes related to the RIN/IRN biosynthesis pathway and starch/sucrose metabolism pathway favored RIN/IRN synthesis. In comparison, the down-regulation of these seven key enzyme genes contributed to the reduction of RIN/IRN. Moreover, the inhibition of photosynthesis is associated with a reduction in RIN/IRN. Photosynthesis was restrained owing to the down-regulation of Lhcb1 and Lhcb6 after 36 mM ethephon treatment and further prevented supply of primary metabolites (such as α-D-glucose) for RIN/IRN synthesis. However, uninterrupted photosynthesis ensured a normal supply of primary metabolites at 18 mM ethephon treatment. AP2/ERF1, bHLH1, and bHLH2 may positively regulate the RIN/IRN accumulation, while NAC1 may play a negative regulatory role. Our results construct the potential bidirectional model for ethylene regulation on RIN/IRN synthesis and provide novel insight into the ethylene-mediated regulation of the metabolism of terpenoid indole alkaloids.

Ginsenoside Rg1 promoted the wound healing in diabetic foot ulcers via miR-489-3p/Sirt1 axis.

PURPOSE: Diabetic foot ulcers (DFUs) are common complications of high severity for diabetes. Ginsenoside Rg1 (Rg1) has the potential for diabetes and cardiovascular diseases therapy. This research aimed at exploring the regulation of Rg1 on DFUs treatment and the underlying mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) incubated with high-glucose culture medium were established for induction of diabetes model. The MTT assay, Annexin V/PI assay and oxidative stress detection were carried out on high-glucose-induced HUVECs. Dual-luciferase reporter assay was performed to prove the interaction of miR-489-3p and Sirt1. DFUs model was established to determine the efficiency of Rg1 and miR-489-3p in wound closure of DFUs in vivo. RESULTS: Rg1 promoted cell proliferation, migration and angiogenesis, and reduced cell apoptosis in high-glucose-induced HUVECs. Knockdown of miR-489-3p alleviated the high-glucose-induced damage to HUVECs, while overexpression of miR-489-3p attenuated the protection effects of Rg1. Overexpression Sirt1 promoted wound healing in DFUs and Sirt1 was a direct target of miR-489-3p. In addition, animal experiments demonstrated that Rg1 promoted wound closure by regulating miR-489-3p/Sirt1 axis. CONCLUSIONS: Rg1 alleviated the DFUs by increasing Sirt1 expression via miR-489-3p downregulation and promoting activation of PI3K/AKT/eNOS signaling.

Knockdown of STIP1 inhibits the invasion of CD133­positive cancer stem­like cells of the osteosarcoma MG63 cell line via the PI3K/Akt and ERK1/2 pathways.

Osteosarcoma is the most common primary malignant tumor of the bone in adolescents and children, with high rates of metastasis and a poor prognosis. Recently, osteosarcoma cancer stem/stem­like cells (CSCs) have been identified as the main cause of recurrence and metastasis. Stress­induced phosphoprotein 1 (STIP1), a co­chaperone that binds to heat shock proteins 70 and 90, is abnormally expressed in several tumor cell lines, and may play an important role in tumor cell migration and invasion. These features indicate that STIP1 may represent a new therapeutic target for osteosarcoma CSCs. However, the role of STIP1 in osteosarcoma CSC migration and invasion remains largely unknown. In the present study, CD133­positive osteosarcoma CSCs were first isolated and cultured by magnetic cell sorting and serum­free medium suspension cell sphere culture, respectively. Knockdown of STIP1 by small interfering RNA significantly was then shown to inhibit the migration and invasion of these cells, possibly due to the regulation of the expression of matrix metalloproteinase (MMP)­2, MMP­9 and tissue inhibitor of metalloproteinase­2. Furthermore, data from the present study suggested that the knockdown of STIP1 decreased the levels of phosphorylated Akt and phosphorylated ERK1/2. In summary, these findings indicate that targeting STIP1 in osteosarcoma may constitute a viable molecular targeted therapy strategy for the inhibition of CSC invasion and migration.

Bone marrow stromal cells-derived exosomes target DAB2IP to induce microglial cell autophagy, a new strategy for neural stem cell transplantation in brain injury.

Bone marrow stromal cells (MSCs) are a useful source of stem cells for the treatment of various brain injury diseases due to their abundant supply and fewer ethical problems compared with transplant treatment. However, the clinical application of MSCs is limited due to allograft rejection and immunosuppression in the process of MSCs transplantation. According to previous studies, microglial cell autophagy occurs following co-culture with MSCs. In the present study, exosomes were obtained from MSCs and subsequently characterized using transmission electron microscopy, atomic force microscopy and dynamic light scattering particle size analysis. The type of microRNAs (miRs) found in the exosomes was then analyzed via gene chip. The results demonstrated that microglial cell autophagy could be induced by exosomes. This mechanism was therefore investigated further via reverse transcription-quantitative PCR, western blotting and luciferase assays. These results demonstrated that exosomes from MSCs could induce microglial cell autophagy through the miR-32-mediated regulation of disabled homolog 2-interacting protein, thus providing a theoretical basis for the clinical application of miRs in MSCs.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.

Effects of long-term rapamycin treatment on glial scar formation after cryogenic traumatic brain injury in mice.

Glial scar impedes axon regeneration and functional recovery following traumatic brain injury (TBI). Although it has been shown that rapamycin (a specific inhibitor of mammalian target of rapamycin) can reduce astrocyte reactivation in the early stage of TBI, its effect on glial scar formation has not been characterized in TBI and other acute brain injury models. To test this, ICR mice received daily administration of rapamycin (0.5 or 1.5 mg/kg, i.p.) beginning at 1 h after cryogenic TBI (cTBI). The results showed that at 3 d post-injury, 1.5 mg/kg rapamycin increased cTBI-induced motor functional deficits and infarct size, and attenuated astrocyte reactivation in the ipsilateral cortex, while 0.5 mg/kg rapamycin did not worsen brain damage and only slightly attenuated astrocyte reactivation. Furthermore, at 7 and 14 d after cTBI, 0.5 mg/kg rapamycin group showed a better motor functional performance than cTBI group. At 14 d post-injury, 0.5 mg/kg rapamycin significantly reduced the area and thickness of glial scar and chondroitin sulfate proteoglycan expression, accompanied by decreased expression of p-S6 and enhanced expression of growth associated protein 43 (an axon regeneration marker) in the region of glial scar. Our data suggest that long-term treatment with rapamycin can inhibit glial scar formation after cTBI, which may be involved in the mechanisms of increased axon regeneration and improved neurological functional recovery, and low-dose rapamycin may be more beneficial for such a therapy.

MiR-214 Regulates the Human Hair Follicle Stem Cell Proliferation and Differentiation by Targeting EZH2 and Wnt/ß-Catenin Signaling Way In Vitro.

miR-214 plays a major role in the self-renewal of skin tissue. However, whether miR-214 regulates the proliferation and differentiation of human hair follicle stem cells (HFSCs) is unknown. Primary HFSCs were isolated from human scalp skin tissue, cultured, and identified using flow cytometry. An miR-214 mimic and inhibitor were constructed for transfection into HFSCs. The MTS and colony formation assays examined cell proliferation. Immunofluorescence detected the localization and expression levels of TCF4, ß-catenin, and differentiation markers. Luciferase reporter and TOP/FOP Flash assays investigated whether miR-214 targeted EZH2 and regulated the Wnt/ß-catenin signaling pathway. Western blot determined the expression levels of enhancer of zeste homolog 2 (EZH2), Wnt/ß-catenin signaling-related proteins, and HFSC differentiation markers in cells subjected to miR-214 transfection. miR-214 expression was remarkably decreased during the proliferation and differentiation of HFSCs into transit-amplifying (TA) cells. Downregulation of miR-214 promotes the proliferation and differentiation of HFSCs. Overexpression of miR-214 led to decreased expression of EZH2, ß-catenin, and TCF-4, whereas downregulation of miR-214 resulted in increased expression of EZH2, ß-catenin, and TCF-4 as well as TA differentiation markers. Immunofluorescence assay revealed that inhibiting miR-214 triggered the entry of ß-catenin and TCF-4 into the nucleus. The luciferase reporter and TOP/FOP Flash assays demonstrated that miR-214 directly targets EZH2 and affects Wnt/ß-catenin signaling. The miR-214/EZH2/ß-catenin axis could be considered a candidate target in tissue engineering and regenerative medicine for HFSCs.

[Vasodilation of quercetin on rat renal artery and the relationship with L-type voltage-gated Ca2+ channels and protein kinase C].

To investigate the diastolic function of quercetin on rat renal artery in vitro and its mechanism, the tension of rat renal artery was recorded by multi myograph system, and the L-type voltage-gated Ca2+ channels (LVGC) current was recorded by whole-cell patch clamp technique. Quercetin produced relaxation effect on rat renal artery pre-contracted by 60 mmol/L KCl or 1 × 10-5 mol/L phenylephrine, and the maximal diastolic percentage was  (84.53 ± 7.35)% or (76.42 ± 4.63)%. There was no statistical difference in the maximal diastolic percentage between endothelium-intact and endothelium-denuded groups. Pre-incubation of protein kinase C (PKC) inhibitor C6303 inhibited the maximal diastolic amplitude induced by quercetin. The peak current density of LVGC in rat renal artery vascular smooth muscle cells (VSMCs) was (23.17 ± 1.33) pA/pF. Quercetin (10 µmol/L) inhibited the peak current to (10.46 ± 1.35) pA/pF, and the inhibition percentage was 54.86%. C6303 (1 µmol/L) partially reversed the inhibitory effect of quercetin, and the inhibition percentage was 62.08% (P < 0.05). These results suggest that quercetin can relax rat renal artery in vitro in a concentration-dependent and endothelium-independent manner. The vasodilation of quercetin may be related to inhibition of LVGC current and activation of PKC.

Activation of caspase-12 at early stage contributes to cardiomyocyte apoptosis in trauma-induced secondary cardiac injury.

Trauma-induced secondary cardiac injury (TISCI) is associated with increased adverse cardiac events and death. We have previously reported that TISCI results in myocardial apoptosis and secondary cardiac dysfunction. However, the underlying mechanism is unclear. To identify the time course of trauma-induced cardiomyocyte apoptosis and possible apoptotic pathway, traumatic rat models were built with Noble-Collip drum. Meanwhile, normal rat cardiomyocytes were cultured with traumatic plasma (TP) for 48 h. Cardiomyocyte apoptosis, cardiac function and the apoptosis related enzymes, including caspase-3, -8, -9, and -12, were determined. The results showed that there was no direct injury of rat hearts immediately after trauma. However, compared with hearts from the sham rats, hearts isolated from traumatic rats exhibited reduced +dP/dTmax and -dP/dTmax 24 h after trauma. In traumatic rats, myocardial apoptotic index and caspase-3 activity obviously increased 6 h after trauma, and achieved the maximal value 12 h after trauma. The activity and expression of caspase-12, an endoplasmic reticulum (ER) stress-specific caspase, elevated markedly 3 h after trauma and reached its peak 6 h after trauma. Otherwise, caspase-8 (extrinsic apoptotic pathway) and caspase-9 (intrinsic apoptotic pathway) in the myocardial tissue of traumatic rats were activated 24 h after trauma. Meanwhile, incubation of normal rat cardiomyocytes with TP increased caspase-12 activity at 6 h, caspase-3 activity at 12 h, caspase-8 and -9 activities at 24 h, respectively. TP-induced cardiomyocyte apoptosis was virtually abolished by Z-ATAD-FMK (a caspase-12 specific inhibitor). In addition, there was a significant negative correlation between myocardial caspase-12 activity and trauma-induced secondary cardiac dysfunction. Our present study demonstrated that caspase-12 is firstly activated and plays an important role in TISCI rats. Inhibition of caspase-12 mediated apoptosis may be a novel strategy in ameliorating posttraumatic cardiomyocyte apoptosis and secondary cardiac injury.

[Mechanisms underlying rat coronary arterial vasoconstriction induced by blockade of inward rectifier potassium channels with BaCl2].

In order to explore the mechanisms underlying the vasoconstriction induced by blockade of inward rectifier K+ channels (Kir) with BaCl2, myogenic tone of isolated rat coronary artery (RCA) was recorded with wire myograph. The dependence of BaCl2- induced contraction on intracellular Ca2+ ([Ca2+]i) release and extracellular Ca2+ ([Ca2+]o) influx was studied by Ca2+ deprivation and restoration. The mechanisms underlying BaCl2-induced RCA contraction were investigated with specific inhibitors. BaCl2 (0.1-1.0 mmol/L) contracted isolated RCA in a concentration-dependent manner and the maximal contraction was (5.69 ± 1.07) mN, nearly equal to contraction induced by 60 mmol/L KCl. The contractions induced by BaCl2 in Ca2+-free solution and by followed restoration of 2.5 mmol/L Ca2+ accounted for (35.44 ± 6.72)% and (64.56 ± 5.94)%, respectively. Calcium channel blocker nifedipine (0.3 µmol/L), cyclooxygenase inhibitor indomethacin (100 µmol/L), ERK1/2 inhibitor PD98059 (10 µmol/L) and chloride channel blocker niflumic acid (100 µmol/L) pretreatment depressed the BaCl2-induced maximal contraction by (87.82 ± 5.43)% (P < 0.01), (73.23 ± 5.47)% (P < 0.01), (75.69 ± 7.94)% (P < 0.01) and (83.24 ± 7.69)% (P < 0.01), respectively. These results demonstrate that BaCl2 induces vasoconstriction in RCA by enhancing both [Ca2+]i release and [Ca2+]o influx, and suggest that increase of prostanoids synthesis, activation of calcium channels and chloride channels, as well as ERK1/2 pathway may be involved in this process.

Efficacy of 50 Hz electromagnetic fields on human epidermal stem cell transplantation seeded in collagen sponge scaffolds for wound healing in a murine model.

To explore the possible efficacy of electromagnetic fields (EMF) for skin tissue engineering, effects of EMF exposure on epidermal stem cells (ESC) seeded in collagen sponge scaffolds for wound healing in a murine model were investigated. The wound models of a full-thickness defect established with 36 7 ∼ 8-week-old nude mice were randomly divided into three groups: a control group, an ESC-only group, and an ESC with EMF exposure group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 20 days). ESC were separated from human foreskin and cultured in vitro, and then transplanted with collagen sponge scaffolds as a delivery vehicle to wounds of the ESC-only group, and ESC with EMF exposure group was exposed to EMF after ESC transplantation. Effects of EMF on morphological changes and expression of ß1 integrin in regenerated skins were observed. Wound healing rates and healing times were collected to evaluate the efficacy of repairment. Results showed that human ESC were successfully transplanted to nude mice, which facilitated the formation of intact skin on nude mice. In contrast to other groups, the wound healing of ESC with EMF exposure group was the fastest (P < 0.05), the structure of regenerated skins was more mature, and it contained more continuity in the number of viable cell layers and rich hair follicles' structure. These results suggest that the use of 50 Hz EMF as a non-invasive treatment can accelerate wound healing of ESC transplantation, and restore structural integrity of regenerated skin. Bioelectromagnetics. 38:204-212,2017. © 2017 Wiley Periodicals, Inc.

Predictive effects of bilirubin on response of colorectal cancer to irinotecan-based chemotherapy.

AIM: To examine the predictive effects of baseline serum bilirubin levels and UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism on response of colorectal cancer to irinotecan-based chemotherapy. METHODS: The present study was based on a prospective multicenter longitudinal trial of Chinese metastatic colorectal cancer (mCRC) patients treated with irinotecan-based chemotherapy (NCT01282658). Baseline serum bilirubin levels, including total bilirubin (TBil) and unconjugated bilirubin (UBil), were measured, and genotyping of UGT1A1*28 polymorphism was performed. Receiver operating characteristic curve (ROC) analysis was used to determine cutoff values of TBil and UBil. The TBil values were categorized into > 13.0 or ≤ 13.0 groups; the UBil values were categorized into > 4.1 or ≤ 4.1 groups. Combining the cutoff values of TBil and UBil, which was recorded as CoBil, patients were classified into three groups. The classifier's performance of UGT1A1*28 and CoBil for predicting treatment response was evaluated by ROC analysis. Associations between response and CoBil or UGT1A1*28 polymorphism were estimated using simple and multiple logistic regression models. RESULTS: Among the 120 mCRC patients, the serum bilirubin level was significantly different between the UGT1A1*28 wild-type and mutant genotypes. Patients with the mutant genotype had an increased likelihood of a higher TBil (P = 0.018) and a higher UBil (P = 0.014) level compared with the wild-type genotype. Patients were stratified into three groups based on CoBil. Group 1 was patients with TBil > 13.0 and UBil > 4.1; Group 2 was patients with TBil ≤ 13.0 and UBil > 4.1; and Group 3 was patients with TBil ≤ 13.0 and UBil ≤ 4.1. Patients in Group 3 had more than a 10-fold higher likelihood of having a response in the simple (OR = 11.250; 95%CI: 2.286-55.367; P = 0.003) and multiple (OR = 16.001; 95%CI: 2.802 -91.371; P = 0.002) analyses compared with the Group 1 individuals. Patients carrying the UGT1A1*28 (TA)7 allele were 4-fold less likely to present with a response compared with the individuals harboring a homozygous (TA)6 genotype in the simple (OR = 0.267; 95%CI: 0.100-0.709; P = 0.008) and multiple (OR = 0.244; 95%CI: 0.088-0.678; P = 0.007) analyses. Classifier's performance of CoBil and UGT1A1*28 were comparable. CONCLUSION: CoBil and UGT1A1*28 are both independent biomarkers for predicting the treatment response of mCRC patients to irinotecan-based chemotherapy. After validation, CoBil, an easily determinable index in the clinic, might be helpful in facilitating stratification of mCRC patients for individualized treatment options.