Neuroprotection of Human Umbilical Cord-Derived Mesenchymal Stem Cells (hUC-MSCs) in Alleviating Ischemic Stroke-Induced Brain Injury by Regulating Inflammation and Oxidative Stress.

Brain injury caused by stroke has a high rate of mortality and remains a major medical challenge worldwide. In recent years, there has been significant attention given to the use of human Umbilical cord-derived Mesenchymal Stem Cells (hUC-MSCs) for the treatment of stroke in different adult and neonate animal models of stroke. However, using hUC-MSCs by systemic administration to treat ischemic stroke has not been investigated sufficiently. In this study, we conducted various experiments to explore the neuroprotection of hUC-MSCs in rats. Our findings demonstrate that an intravenous injection of a high dose of hUC-MSCs at 2 × 10^7 cells/kg markedly ameliorated brain injury resulting from ischemic stroke. This improvement was observed one day after inducing transient middle cerebral artery occlusion (MCAO) and subsequent reperfusion in rats. Notably, the efficacy of this single administration of hUC-MSCs surpassed that of edaravone, even when the latter was used continuously over three days. Mechanistically, secretory factors derived from hUC-MSCs, such as HGF, BDNF, and TNFR1, ameliorated the levels of MDA and T-SOD to regulate oxidative stress. In particular, TNFR1 also improved the expression of NQO-1 and HO-1, important proteins associated with oxidative stress. More importantly, TNFR1 played a significant role in reducing inflammation by modulating IL-6 levels in the blood. Furthermore, TNFR1 was observed to influence the permeability of the blood-brain barrier (BBB) as demonstrated in the evan's blue experiment and protein expression of ZO-1. This study represented a breakthrough in traditional methods and provided a novel strategy for clinical medication and trials.

Effects of different concentrations of mesenchymal stem cells treatment on LPS-induced acute respiratory distress syndrome rat model.

PURPOSE: This study was prospectively designed to investigate the effects of different concentrations of mesenchymal stem cells treatment on respiratory mechanics, oxygenation, hemodynamics and inflammatory response in LPS-induced acute respiratory distress syndrome (ARDS) rat model. Methods: One hundred and twenty six LPS-induced ARDS model rats (weighted 200-220 g) were randomly divided into three groups: 1) Control group (N = 42); 2) low-dose hUC-MSC treatment group (MSC group 1, 1x107 cell/kg, N = 42); 3) high-dose hUC-MSC treatment group (MSC group 2, 2x107 cell/kg, N = 42), sham operation group as healthy group (N = 15). The rats were observed closely for 24 hours after hUC-MSC treatment, and the survival rate was calculated. At 24 hours, all rats were tested for hemodynamics, blood gas analysis, heart, lung, liver and kidney functions, inflammatory factors detection in blood samples and broncho-alveolar lavage fluid (BALF). The lung tissue of the rats was collected for HE staining analysis. Results: After LPS injection, ARDS was obvious in all LPS-infused rat groups, consistent with severe acute lung injury and high death rate. However, compared with the control group, a single intravenous injection hUC-MSC at dose of 1 × 107 cells/kg (low dose group) and 2 × 107 cells/kg (high dose group) reduced the mortality of rats with LPS-induced ARDS, as well as improving the lung function, increased the arterial oxygen pressure, improved the heart function, and reduced the levels of inflammatory factors including IL-1ß, IL-6, and TNF-α. In addition, the high dose MSC group showed better lung injury therapeutic effects than the low dose MSC group. Data from this study demonstrated that injection of hUC-MSC had a significant therapeutic effect in treating the rat model of LPS-induced ARDS and multiple organ function injury.

Recombinant Neorudin for the Prevention of Deep-Vein Thrombosis After Spinal-Cord Injury.

Background: Whether anticoagulant therapy should be used after spinal-cord injury (SCI) surgery was controversial. The anticoagulation characteristics of a newly developed anticoagulant, recombinant neorudin (EPR-hirudin (EH)), were explored using a rat model of SCI to provide a basis for clinical anticoagulation therapy of SCI. Methods: A rat model of SCI was developed by Allen's method. Then, thrombosis in the inferior vena cava was induced by ligation. The low-bleeding characteristics of EH were explored by investigating dose-response and time-effect relationships, as well as multiple administration of EH, on thrombus formation complicated with SCI. Results: EH inhibited thrombosis in a dose-dependent manner by reducing the wet weight and dry weight of the thrombus. An inhibiting action of EH on thrombosis was most evident in the group given EH 2 h after SCI. After multiple intravenous doses of EH, thrombosis inhibition was improved to that observed with low molecular weight heparin (LMWH) (87% vs 90%). EH administration after SCI neither increased bleeding in the injured spine nor damaged to nerve function. Bleeding duration and activated partial thromboplastin time were increased in the high-dose EH group compared with that in the normal-saline group, but were lower than those in the LMWH group. Conclusion: EH can reduce thrombus formation in a rat model of SCI, and bleeding is decreased significantly compared with that using LMWH. EH may prevent thrombosis after SCI or spinal surgery.

The Antithrombotic Effect of Recombinant Neorudin on Thrombi.

Introduction: Recombinant neorudin (EPR-hirudin, EH) was developed through the addition of an EPR (Glu-Pro-Arg) peptide to the amino terminus of hirudin, which can be recognized and cut by coagulation factors XIa (FXIa) and/or Xa (FXa). In this study, the low-bleeding antithrombotic effects of EH were evaluated utilizing experimental models of thrombosis in rabbits and rats to provide a test basis for clinical trials. Methods: The bleeding risks of EH and hirudin were first compared in mice by the tail-clipping method, and then the antithrombotic activity of EH was investigated in a rabbit model of arteriovenous bypass thrombosis and a rat model of thrombotic cerebral infarction. Results: In mice, intravenous administration of EH at 1.5 mg/kg and 3 mg/kg did not affect the bleeding time compared with normal saline, while the administration of hirudin at 1.5 mg/kg prolonged the bleeding time by over 3 times the administration of normal saline. Furthermore, intravenous administration of EH had a significant dose-dependent inhibitory effect on the formation and development of arteriovenous bypass thrombosis and thrombotic cerebral infarction. Compared with an equimolar dose of hirudin, the antithrombotic effect of EH was similar, while the bleeding side effects were significantly attenuated. Moreover, when the antithrombotic effects were similar, EH had a shorter bleeding time and was associated with less bleeding than low molecular weight heparin (LMWH). EH had a therapeutic effect on thrombotic cerebral infarction without increasing the occurrence of cerebral hemorrhage. Conclusion: The findings from the preclinical animal models used in this study showed that EH could not only effectively inhibit thrombus formation but also reduce the risk of bleeding.

Therapeutic Efficacy of Human Mesenchymal Stem Cells With Different Delivery Route and Dosages in Rat Models of Spinal Cord Injury.

Recent studies have shown that the use of mesenchymal stem/stromal cells (MSCs) may be a promising strategy for treating spinal cord injury (SCI). This study aimed to explore the effectiveness of human umbilical cord-derived MSCs (hUC-MSCs) with different administration routes and dosages on SCI rats. Following T10-spinal cord contusion in Sprague-Dawley rats (N = 60), three different dosages of hUC-MSCs were intrathecally injected into rats (SCI-ITH) after 24 h. Intravenous injection of hUC-MSCs (SCI-i.v.) and methylprednisolone reagent (SCI-PC) were used as positive controls (N = 10/group). A SCI control group without treatment and a sham operation group were injected with Multiple Electrolyte Injection solution. The locomotor function was assessed by Basso Beattie Bresnahan (BBB) rating score, magnetic resonance imaging (MRI), histopathology, and immunofluorescence. ELISA was conducted to further analyze the nerve injury and inflammation in the rat SCI model. Following SCI, BBB scores were significantly lower in the SCI groups compared with the sham operation group, but all the treated groups showed the recovery of hind-limb motor function, and rats receiving the high-dose intrathecal injection of hUC-MSCs (SCI-ITH-H) showed improved outcomes compared with rats in hUC-MSCs i.v. and positive control groups. Magnetic resonance imaging revealed significant edema and spinal cord lesion in the SCI groups, and significant recovery was observed in the medium and high-dose hUC-MSCs ITH groups. Histopathological staining showed that the necrotic area in spinal cord tissue was significantly reduced in the hUC-MSCs ITH-H group, and the immunofluorescence staining confirmed the neuroprotection effect of hUC-MSCs infused on SCI rats. The increase of inflammatory cytokines was repressed in hUC-MSCs ITH-H group. Our results confirmed that hUC-MSC administered via intrathecal injection has dose-dependent neuroprotection effect in SCI rats.

Corrigendum to "Effects of total iridoid glycosides of Picrorhiza scrophulariiflora against non-alcoholic steatohepatitis rats induced by high-fat and high-sugar diet through regulation of lipid metabolism" [Chin Herb Med 12 (2019) 67-72].

[This corrects the article DOI: 10.1016/j.chmed.2019.12.005.].

Corrigendum to "Neuroprotective effect of Sanqi Tongshuan Tablets on sequelae post-stroke in rats" [Chinese Herbal Medicines 11 (2019) 45-51].

[This corrects the article DOI: 10.1016/j.chmed.2018.12.002.].

Effects of total iridoid glycosides of Picrorhiza scrophulariiflora against non-alcoholic steatohepatitis rats induced by high-fat and high-sugar diet through regulation of lipid metabolism.

Objective: To investigate the therapeutic effect of total iridoid glycosides of Picrorhiza scrophulariiflora (TIGP) on non-alcoholic steatohepatitis (NASH). Methods: SD rats were fed with high-fat and high-sugar diet for 8 weeks to establish NASH. TIGP were given orally at doses of 20, 40 and 80 mg/kg/d for 4 weeks. Triglycerides assay (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose (FPG), fasting insulin (FINS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), chemokine-1 (MCP-1), leptin (LEP) in serum were tested. TG, TC, superoxide dismutase (SOD), malondialdehyde (MDA), and free fatty acid (FFA) in liver tissue were determined by colorimetric methods. Steatosis of hepatocytes and inflammation was performed by pathological examination. Results: The results showed that TIGP significantly decreased TC, TG and FFA in liver tissue, increased SOD activity, decreased MDA content, decreased serum levels of TG, TC, HDL-C/LDL-C, ALT, AST, GLU, HOMA-IR, TNF-α and LEP, and in addition, improved steatosis of liver cells compared to NASH. Conclusion: TIGP had anti-fatty liver effect against NASH rats induced by high-fat and high-sugar diet. Its mechanism was related to the regulation of lipid metabolism and reduction of insulin resistance, through inhibition of oxidative stress and inflammation.

Thrombolysis with rhPro-UK 3 to 6 hours after embolic stroke in rat.

Objectives: To investigate the thrombolysis with recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rats at different therapeutic time windows (TTW). Methods: Rats were subjected to embolic middle cerebral artery occlusion. RhPro-UK and positive control drugs rt-PA,UK were administered 3 h, 4.5 h, 6 h after inducing thromboem-bolic stroke. Neurological deficit scoring (NDS) was evaluated at 6 h and 24 h after the treatment. The lesion volume in cerebral hemispheres was measured by MRI scanning machine after 6 h of thrombolysis, and the infarct volume was measured by TTC stain, together with hemorrhagic volume quantified by a spectrophotometric assay after 24 h of thrombolysis. Results: RhPro-UK 10, 20 × 104 U/kg significantly improved the NDS after cerebral thromboembolism in rats at 3 h, 4.5 h TTW, and at the 6 h TTW, the NDS was improved by 28.0% (P = 0.0690) and 29.2% (P = 0.0927) at 6 h and 24 h after rhPro-UK 20 ×104 U/kg administration, respectively. RhPro-UK 10, 20 × 104 U/kg significantly reduced the brain lesions measured by MRI at 3 h and 4.5 h TTW. RhPro-UK 10, 20 × 104 U/kg significantly reduced the cerebral infarction measured by TTC at 3 h, 4.5 h TTW. There was no increase in cerebral hemorrhage compared with untreated group after rhPro-UK administration. Conclusions: RhPro-UK had an obvious therapeutic effect on ischemic stroke caused by thrombosis, and could be started within 4.5 h TTW with less side effects of cerebral hemorrhage than that of UK.

Effect of recombinant human prourokinase on thrombolysis in a rabbit model of thromboembolic stroke.

The aim of the present study was to investigate the efficacy of recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rabbits. A total of 210 rabbits were used in experiments. The 180 thromboembolic stroke rabbits were divided into three therapeutic time windows with six groups in each time window (n=10). The model group was administered saline, the reagent groups were administered rhPro-UK (2.5×, 5× and 10×104 U/kg), and the positive control groups were administered 5×104 urokinase (UK) U/kg and 4.5 mg/kg recombinant human tissue plasminogen activator via intravenous infusion at 3, 4.5 and 6 h after embolism. The remaining 30 rats (that had not undergone occlusion by autologous blood clots) served as a sham group and were administered saline. The radioactive intensity was detected using a medical gamma counter before and after the administration of the drug for 15, 30, 45, 60, 75, 90, 105 and 120 min. At 24 h after treatment, the brain samples were coronally sliced into 5 mm sections and hemorrhage was estimated used a semiquantitative method by counting the number of section faces with hemorrhaging. The plasma was collected for prothrombin time, activated partial thromboplastin time, fibrinogen and thrombin time tests using a solidification method with a blood coagulation factor analyzer. In addition, α2-antiplasmin (α2-AP) was evaluated using ELISA methods using a RT-6100 microplate reader. At the 3 h time point, the thrombolysis rate of rhPro-UK(2.5×, 5× and 10×104 U/kg) was 21.5% (P<0.05), 36.8% (P<0.001) and 55.0% (P<0.001), respectively together with patency rates of 10% (P>0.05), 40% (P<0.05) and 70% (P<0.001). Furthermore, α2-AP levels were reduced by 5.3% (P>0.05), 5.3% (P>0.05) and 18.1% (P<0.05). At the 4.5 h time point, the thrombolysis rate was 18.8% (P<0.05), 29.9% (P<0.01) and 49.0% (P<0.001) together with patency rates of 10% (P>0.05), 30% (P<0.05) and 50% (P<0.01), and α2-AP levels were reduced by 2.4% (P>0.05), 6.5% (P>0.05) and 17.8% (P<0.05). At the 6 h time point, the thrombolysis rate was 14.7% (P<0.05), 24.1%(P<0.01) and 35.7% (P<0.001) together with patency rates of 20% (P>0.05), 30% (P<0.05) and 40% (P<0.01), and α2-AP levels were reduced by 5.7% (P>0.05), 12.7% (P>0.05) and 22.2% (P<0.01). No significant differences (P>0.05) were identified between rhPro-UK (2.5×, 5× and 10×104 U/kg) and the model group regarding hemorrhage type, size and blood coagulation factors at the different time points. Thus, rhPro-UK promoted thrombolysis and recanalization (patency rate), with reduced risk of cerebral hemorrhage, and thus exerted protective effects on cerebral ischemia rabbits.

Transcriptomic Study Reveals Recovery of Impaired Astrocytes Contribute to Neuroprotective Effects of Danhong Injection Against Cerebral Ischemia/Reperfusion-Induced Injury.

Danhong Injection (DHI) is widely used in clinics for treating cardiovascular and cerebrovascular diseases in China. However, the mode of action of DHI for neuroprotection remains unclear. In the present study, we deemed to investigate the effects of DHI on a rat model of cerebral ischemia/reperfusion injury (IRI) with an emphasis on its regulated gene profile obtained from microarray assays. Firstly, we showed that a 14-day DHI treatment effectively ameliorated severity of neurological deficits, reduced size of ischemic damage, improved status of oxidation stress, as well as systemic inflammation for IRI rats, along with which was a pronounced reduced cell infiltration in the area of periaqueductal gray matter. Secondly, bioinformatic analyses for the 429 differentially expressed genes (DEGs) regulated by DHI treatment pointed out ECM-receptor interaction, neuroactive ligand-receptor interaction, and endocytosis as the top three biological processes, while Toll-like recptor 4 (TLR4) as the most relavant singaling molecule. Lastly, we provided evidences showing that DHI might directly protect primary astrocytes from oxygen and glucose deprivation/re-oxygenation (OGD/Re) injury, the effects of which was associated with LAMC2 and ADRB3, two DEGs related to the top three biological processes according to transcriptomic analysis. In conlusion, we reported that DHI might work through maintaining the integrity for brain-blood barrier and to regulate TLR4-related signaling pathway to diminish the inflammation, therefore, effectively improved the outcomes of IRI. Our findings suggested that the attenuated astrocytic dysfunction could be a novel mechanism contributing to the neuroprotective effects of DHI against cerebral ischemia/reperfusion-induced damage.

Effect of human recombinant prourokinase(rhpro-UK) on thromboembolic stroke in rats.

We evaluated the efficacy and safety of human recombinant prourokinase ( rhpro-UK) on thromboembolic stroke in rats. 60 rats with thromboembolic stroke were divided into 6 groups (n = 10). The model group was given saline, the reagent groups were given rhpro-UK (5, 10, 20 × 104U/kg), and positive control groups were given urokinase (UK) 10 × 104U/kg and recombinant tissue plasminogen activator (rt-PA) 9mg/kg through intravenous infusion at 1.5h after embolism. And other 10 rats without occluded by autologous blood clots as the sham group were given saline. At 6h after treatment, neurological deficit score and Magnetic Resonance Imaging(MRI) including T1WI and T2WI sequence scanning were measured. At 24h after treatment, the brain was cut for 2,3,5-triphenyltetrazolium chloride (TTC) staining and aspectrophotometric assay to measure the infarct area and intracerebral hemorrhage after neurological deficit detection. rhpro-UK (5, 10, 20 × 104 U/kg) improved neurological disorder by 39.1 ± 19.7% (n = 10, P > 0.05), 65.2 ± 14.2% (n = 10, P < 0.01) and 65.2 ± 14.2% (n = 10, P < 0.01) maximally; decreased brain lesion volume by 36.7 ± 34.8% (n = 10, P < 0.05), 77.6 ± 7.7% (n = 10, P < 0.01) and 80.5 ± 6.9% (n = 10, P < 0.01); decreased infarction area by 38.2 ± 24.0% (n = 10, P < 0.01), 73.9 ± 5.2% (n = 10, P < 0.001) and 79.7 ± 4.0% (n = 10, P < 0.001) respectively, and there were no statistics difference between rhpro-UK (5, 10, 20 × 104 U/kg) and each positive groups at intracerebral hemorrhage (P > 0.05). Rhpro-UK improved the damaged neural function, decreased the extent of the disease and did not raise bleeding, had protective effects for cerebral ischemia in rats.