Rat Cells Research Articles

Novel Therapeutic Potential of 'Ambon' Banana and Green Bean Sprout Extracts in Mitigating Cigarette Smoke-Induced Testicular Oxidative Stress and Sperm Quality Impairment

Cigarette smoke exposure generates free radicals, causing oxidative stress and adversely affecting reproductive organs, resulting in reduced sperm quality. The aim of this study was to investigate the therapeutic benefits of 'Ambon' banana and green bean sprout extracts, either individually or in combination, in mitigating testicular MDA levels and preserving spermatogenic cells in rats exposed to cigarette smoke. 35 Male rats, aged 3 months, were divided into five groups: negative control, positive control (cigarette smoke-exposed), P1 (cigarette smoke + 'Ambon' banana extract), P2 (cigarette smoke + green bean sprout extract), and P3 (cigarette smoke + 'Ambon' banana and green bean sprout extracts). Cigarette smoke exposure was administered at 4 sticks/day/group for 30 days, and extracts were orally administered using a sonde for the same duration. The study showed that 'Ambon' banana extract, green bean sprout extract, and their combination significantly reduced testicular MDA levels, increased the number of spermatogonia, spermatocytes, spermatids, testicular weight, diameter of seminiferous tubules, and the number of mouse Leydig cells in rats. This study highlights the potential therapeutic effects of 'Ambon' banana and green bean sprout extracts as protective agents against cigarette smoke-induced testicular oxidative stress and sperm quality impairment.

Synthesis and biological evaluations of 8-biaryl-2,2-dimethylbenzopyranamide derivatives against Alzheimer's disease and ischemic stroke

Alzheimer's disease, the commonest cause of dementia, is a growing global health concern with huge implications for individuals and society. Stroke has still been a significant challenge in clinics for a long time, which is the second leading cause of death in the world, especially ischemic stroke. Both Alzheimer's disease and stroke are closely related to oxidative stress and HIF-1 signaling pathways in nerve cells. Herein, we describe our structure-based design, synthesis, and biological evaluation of a new class of 8-biaryl-2,2-dimethylbenzopyranamide derivatives as natural product derivatives. Our efforts have resulted in the discovery of highly potent neuroprotective agents, as exemplified by compound D13 as a HIF-1α inhibitor, which significant improvement in the behavior of Alzheimer's disease mice and shows great potential improvement of brain infarct volume in pMCAO model rats, improves the increase of blood–brain barrier permeability after cerebral ischemia in rats, neuroprotective effect, reduce the level of apoptotic cells in rats after cerebral ischemia, better than Edaravone.

Papaya Leaf Ethanol Extract Lowers Malondialdehyde Levels, Increases Superoxide Dismutase Levels, and Increases Leydig Cell Count in Male Rats Given High-Intensity Swimming Exercise

Physical activity is any body movement that occurs due to skeletal muscle contractions that require energy expenditure. Overtraining can trigger oxidative stress due to chronic systemic hypoxia, cause an increase in systemic inflammatory response and multiorgan damage, and accelerate the aging process. The aging process is influenced by many factors, one of which is high-intensity swimming exercise which triggers the emergence of free radicals and leads to systemic oxidative stress. High-intensity swimming exercise is associated with an increase in Reactive Oxygen Species (ROS). The Papaya plant is famous for its therapeutic and nutritional properties. Papaya leaves contain secondary metabolites from papaya leaf extract including tannins, saponins, flavonoids, phenolics, and alkaloids. Papaya leaf extract has strong medicinal properties as an antibacterial, antiviral, antitumor, and antioxidant. This research is an experimental study with a post-test-only control group design. The sample in this study were male white rats (Rattus norvegicus) with an age range of 3-4 months with an average body weight range of 180-200 grams. As many as 36 rats were divided into 2 treatment groups. Treatment group 1 (P1) rats were given high-intensity swimming exercise treatment and aquabidest. Treatment group 2 (P2) rats were given high-intensity swimming exercise treatment and papaya leaf extract 60mg / 200g BB orally. Each treatment was carried out for 21 days. The data obtained will be analyzed using an independent sample T-test. The results of this study obtained: (1) the average MDA levels showed that the P1 group (1.02±0.064) was higher than the P2 group (0.86±0.44) with a value of p<0.001; (2) the average SOD level in the P2 group (2.72±0.437) was higher than P1 (1.46±0.288) with p<0.001; (3) the average number of Leydig cells in the P1 group (13.0±5.05) was lower than in the P2 group (30.0±5.05) with p<0.001. This study concluded that papaya leaf ethanol extract in rats was able to reduce MDA levels, increase SOD levels and increase the number of Leydig cells in male rats given high-intensity swimming exercise.

Effectiveness of Avocado Seed Extract (Persea Americana Mill) in Maintaining Fibroblast Cell Viability of Avulsed Teeth in Male Wistar Rats

Background: Avulsion is a complex trauma characterized by the loss of a tooth from its socket. An avulsion condition requires immediate replantation to return the tooth to its socket. The success of this action is influenced by the viability of the fibroblast cells. One of the measures to maintain viability is to store avulsed teeth in storage media. Avocado seed (Persea Americana Mill) is a plant that is easily available and contains flavonoids and saponins which have anti-inflammatory, antioxidant and antibacterial activities. Avocado seed extract is known to play a role in wound healing by increasing the number of fibroblast cells. Objective: to determine the effectiveness of avocado seed extract (Persea Americana Mill) in maintaining the viability of avulsed tooth fibroblast cells in male Wistar rats. Methods: The sample used was 24 Wistar rat teeth extracted and divided into 2 groups, 1 control group and 1 treatment group, where each group was divided into 2 subgroups based on soaking time, namely 30 and 60 minutes. Furthermore, fibroblast cells in the periodontal ligament are trypsinated and then counted with a hemocytometer on a light microscope. The data were then tested using SPSS 24 software. Results: There was a significant difference in the amount of fibroblast cell viability in immersion using avocado seed extract and HBSS. Conclusion: Soaking using HBSS for 30 minutes produced the best fibroblast cell viability.

N-Acetylserotonin Alleviates Retinal Autophagy via TrkB/AKT/Nrf2 Signaling Pathway in Retinal Ischemia-Reperfusion Injury Rats

Introduction: The objective of this study was to investigate the impact of N-acetylserotonin (NAS) on the autophagy of retinal cells in rats with retinal ischemia-reperfusion injury (RIRI) and to explore the mechanisms by which NAS administration can alleviate RIRI through the tropomyosin-related kinase receptor B (TrkB)/protein kinase B (Akt)/nuclear factor erythroid-derived factor 2-related factor (Nrf2) signaling pathway. Methods: Healthy adult male rats were randomly assigned to four groups: sham, RIRI, RIRI+NAS, and RIRI+NAS+ANA-12. The RIRI group was induced by elevating intraocular pressure, and changes in retinal structure and edema were assessed using H&E staining. The RIRI+NAS and RIRI+NAS+ANA-12 groups received intraperitoneal injections of NAS before and after modeling. The RIRI+NAS+ANA-12 group was also administered ANA-12, a TrkB antagonist. Immunohistochemical staining and Western blot analysis were used to evaluate phosphorylated TrkB (p-TrkB), phosphorylated Akt (p-Akt), Nrf2, sequestosome 1 (P62), and microtubule-associated protein 1 light chain 3 (LC3-II) levels in the retinas of each group. Electroretinogram was recorded to detect retinal function in each group of rats 24 h after modeling. Results: The RIRI+NAS group had a thinner retina and more retinal ganglion cells (RGCs) than RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Immunohistochemical staining and Western blot results showed that p-TrkB, p-Akt, n-Nrf2, and P62 levels in the RIRI+NAS group were higher compared with those in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Also, lower LC3-II levels were observed in the RIRI+NAS group compared with that in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Electroretinogram recording results showed that 24 h after retinal ischemia-reperfusion, the magnitude of b-wave changes was attenuated in the RIRI+NAS group compared with the RIRI group (p < 0.05). Conclusion: The administration of NAS activates the TrkB/Akt/Nrf2 signaling pathway, reduces autophagy, alleviates retinal edema, promotes the survival of retinal ganglion cells (RGCs), and provides neuroprotection against retinal injury.

Effect of mesenchymal stem cells on animal semen during storage

Mesenchymal stem cells (MSCs) have been known to mankind since the mid-20th century. The comprehensive study revealed their high biologically active potential. Capacity of forming several types of body tissues was demonstrated. The stem cells, like any other cells, exert their effect on surrounding cells and tissues by secreting extracellular vesicles. The extracellular vesicles of the stem cells possess biological activity of parent cells. Taking into account the regenerative potential of the mesenchymal stem cells, they are currently used in medicine, and also in veterinary medicine for treatment of various injuries of the companion animals. Effect of the mesenchymal stem cells on boar and rat sperm cells during 12-hour storage was studied. The study results demonstrated that during 12 hours of coincubation, the porcine MSCs contributed to the survival of the boar sperm cells and maintenance of their motility at 60–80% (depending on the solvent) as compared to the controls. Such a significant effect was not however observed during coincubation of the rat sperm cells with rat MSCs. But it should be noted that before the3rdhour of coincubation, the experimental sperm motility was higher than that of the control. By hour5 of the observation, this difference was leveled. The rat and boar sperm cells are likely to have different physiological characteristics, which were reflected in the results obtained. Therefore, possibility of using the MSCs for the storage and cryopreservation of the semen of some animals was demonstrated, but this requires further research.

Probiotics Attenuate Myopathic Changes in Aging Rats via Activation of the Myogenic Stellate Cells.

Aging represents a complex biological process associated with decline in skeletal muscle functions. Aging impairs satellite cells that serve as muscle progenitor cells. Probiotic supplementation may have many beneficial effects via various mechanisms. We examined the possible effects of probiotics in stimulating the proliferation of myogenic stellate cells in aging rats. Twenty-four male albino Sprague-Dawley rats were classified equally into four groups: adult control, old control, adult + probiotics, and old + probiotics. Probiotics (Lactobacillus LB) were administered gavage at a dose of 1ml (1 × 109CFU/ml/day) for 4 weeks. A significant increase in the relative gastrocnemius weight ratio and improvement of contractile parameters was detected in the old + probiotics group (0.6 ± 0.01) compared to the old control group (0.47 ± 0.01; P < 0.001). Probiotics significantly upregulated the activities of GSH, while NO and MDA were markedly decreased compared to control groups (P ≤ 0.001). Also, probiotics increased the mRNA and protein expressions of myogenin and CD34 (P < 0.05) as determined by real-time PCR and immunohistochemistry. Moreover, the old + probiotics group showed apparent restoration of the connective tissue spaces, reflecting the all-beneficial effects of probiotics. Our findings indicated that probiotics attenuated myopathic changes in aging rats probably through activation of the myogenic stellate cells. Probiotics improved the muscle weight, function, antioxidant activity, and myogenic transcription factors of the skeletal muscle.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Bardoxolone Methyl Ameliorates Chemotherapy-Induced Neuropathic Pain by Activation of Phosphorylated Nuclear Factor Erythroid 2-Related Factor 2 in the Dorsal Root Ganglia.

Many chemotherapeutic drugs, including paclitaxel, produce neuropathic pain in patients with cancer, which is a dose-dependent adverse effect. Such chemotherapy-induced neuropathic pain (CINP) is difficult to treat with existing drugs. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major regulator of antioxidative responses and activates phosphorylated Nrf2 (pNrf2). We determined the analgesic effects of bardoxolone methyl (BM), an Nrf2 activator, and the role of pNrf2 on CINP. CINP was induced in rats by intraperitoneally injecting paclitaxel on 4 alternate days in rats. BM was injected systemically as single or repeated injections after pain fully developed. RNA transcriptome, mechanical hyperalgesia, levels of inflammatory mediators and pNrf2, and location of pNrf2 in the dorsal root ganglia (DRG) were measured by RNA sequencing, von Frey filaments, Western blotting, and immunohistochemistry in rats and human DRG samples. In addition, the mitochondrial functions in 50B11 DRG neuronal cells were measured by fluorescence assay. Our RNA transcriptome of CINP rats showed a downregulated Nrf2 pathway in the pain condition. Importantly, single and repeated systemic injections of BM ameliorated CINP. Paclitaxel increased inflammatory mediators, but BM decreased them and increased pNrf2 in the DRG. In addition, paclitaxel decreased mitochondrial membrane potential and increased mitochondrial volume in 50B11 cells, but BM restored them. Furthermore, pNrf2 was expressed in neurons and satellite cells in rat and human DRG. Our results demonstrate the analgesic effects of BM by Nrf2 activation and the fundamental role of pNrf2 on CINP, suggesting a target for CINP and a therapeutic strategy for patients.

In Silico, In Vitro, and In Vivo Evaluation of Caffeine-Coated Nanoparticles as a Promising Therapeutic Avenue for AML through NF-Kappa B and TRAIL Pathways Modulation.

Advancements in nanoscience have led to a profound paradigm shift in the therapeutic applications of medicinally important natural drugs. The goal of this research is to develop a nano-natural product for efficient cancer treatment. For this purpose, mesoporous silica nanoparticles (MSNPs) were formulated, characterized, and loaded with caffeine to develop a targeted drug delivery system, i.e., caffeine-coated nanoparticles (CcNPs). In silico docking studies were conducted to examine the binding efficiency of the CcNPs with different apoptotic targets followed by in vitro and in vivo bioassays in respective animal models. Caffeine, administered both as a free drug and in nanomedicine form, along with doxorubicin, was delivered intravenously to a benzene-induced AML model. The anti-leukemic potential was assessed through hematological profiling, enzymatic biomarker analysis, and RT-PCR examination of genetic alterations in leukemia markers. Docking studies show strong inter-molecular interactions between CcNPs and apoptotic markers. In vitro analysis exhibits statistically significant antioxidant activity, whereas in vivo analysis exhibits normalization of the genetic expression of leukemia biomarkers STMN1 and S1009A, accompanied by the restoration of the hematological and morphological traits of leukemic blood cells in nanomedicine-treated rats. Likewise, a substantial improvement in hepatic and renal biomarkers is also observed. In addition to these findings, the nanomedicine successfully normalizes the elevated expression of GAPDH and mTOR induced by exposure to benzene. Further, the nanomedicine downregulates pro-survival components of the NF-kappa B pathway and upregulated P53 expression. Additionally, in the TRAIL pathway, it enhances the expression of pro-apoptotic players TRAIL and DR5 and downregulates the anti-apoptotic protein cFLIP. Our data suggest that MSNPs loaded with caffeine, i.e., CcNP/nanomedicine, can potentially inhibit transformed cell proliferation and induce pro-apoptotic TRAIL machinery to counter benzene-induced leukemia. These results render our nanomedicine as a potentially excellent therapeutic agent against AML.

Structure-based design, and development of amidinyl, amidoximyl and hydroxamic acid based organic molecules as novel antimalarial drug candidates

Malaria remains a significant global health concern causing numerous fatalities and the emergence of antimalarial drug resistance highlights the urgent need for novel therapeutic options with innovative mechanisms of action and targets. This study aimed to design potential inhibitors of Plasmodium falciparum 6-pyruvoyltetrahydropterin synthase (PfPTPS), synthesize them, and experimentally validate their efficacy as antimalarial agents. A structure-based approach was employed to design a series of novel derivatives, including amidinyl, amidoximyl and hydroxamic acid analogs (1c, 1d, 2b, and 3b), with a focus on their ability to bind to the Zn2+ present in the active site of PfPTPS. The syntheses of these compounds were accomplished through various multi-step synthetic pathways and their structural identities were confirmed using 1H and 13C NMR spectra, mass spectra, and elemental analysis. The compounds were screened for their antiplasmodial activity against the NF54 strain of P. falciparum and in vitro cytotoxicity testing was performed using L-6 cells. The in vivo acute toxicity of the compounds was evaluated in mice. Docking studies of the compounds with the 3D structure of PfPTPS revealed their strong binding affinities, with compound 3b exhibiting notable metal–acceptor interaction with the Zn2+ in the protein binding pocket thereby positioning it as a lead compound for PfPTPS inhibition. The in vitro antiplasmodial studies revealed moderate efficacies against the Pf NF54 strain, particularly compounds 1d and 3b which displayed IC50 < 0.2 μM. No significant cytotoxicity was noted on the L-6 rat cell line. Moreover, in vivo studies suggested that compound 3b exhibited both safety and efficacy in treating rodent malaria. The identified lead compound in this study represents a possible candidate for antimalarial drug development and can be further explored in the search for alternative antifolate drugs to combat the malaria menace.

Recording changes in biochemical parameters &lt;i&gt;in vivo&lt;/i&gt; in the ischemic stroke model

Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues. In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University. The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions. Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.

Quality Evaluation of Malted Pigeon Pea –Orange Fleshed Sweet Potato Flour Blends and Biscuits Using Albino (Wistar) Rats

The use of underutilized food crops and their fortification could add value to food products. Malted pigeon pea was produced and substituted into orange fleshed sweet potato at 0, 5, 10, 15, 20, and 100% to produce flour blends which was used to feed albino rats on addition of salt, bone meal, blood meal. The blend flour was also used to produce biscuits on addition of baking fat, sugar and salt. The biscuit was analysed for physical and sensory properties. The albino rat feed was analysed for nutrition and haematological qualities. The ash, protein and energy value content of the flour increased from 2.34 to 3.11, 4.64 to 13.27%, 379.83 to 384.19 Kcal/100g, respectively, with increase in added malted pigeon flour. The flavonoid, saponin, phenol, phytate, tannins contents increased from 2.11 to 3.43, 6.61 to 9.70, 0.03 to 0.07, 33.74 to 50.28, 0.04 to 0.08 mg/kg, respectively. The water absorption capacity and bulk density capacity decreased from 0.38 to 0.15% and 0.73 to 0.46 g, while the swelling, oil absorption and foam capacity increased from 1.60 to 3.65, 0.91 to 1.82 and 7.15 to 12.20%, respectively, with addition of malted pigeon pea. The highest break strength was observed in biscuit produced with 85:15 (85 % orange fleshed sweet potato, 15 % malted pigeon pea flour blends) (3000g). The Feed Efficient Ratio (FER) and the Protein Efficient Ratio (PER) of the feed albino rats improved with addition of malted pigeon pea. The Haemoglobin (HB), Park Cell Volume (PCV), Total White Blood Cells (TWBC), White Blood Cells (WBC), Red Blood Cell (RBC) of the feed albino rats generally increased with increase in the quantity of malted pigeon pea over the three weeks feeding duration. All the blend flour biscuits were generally accepted; however, the 15% malted pigeon pea biscuit was the most preferred. Quality and acceptable biscuits can be produced from malted pigeon pea –orange fleshed sweet potato.

Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

Investigation on the effect of ulinastatin on the apoptosis of vascular smooth muscle cells in rats with aortic dissection based on the Sirt1/FoxO3a pathway.

This study aimed to investigate the effects of ulinastatin on the apoptosis and (Sirt1/FoxO3a) pathway of vascular smooth muscle cells (VSMC) in aortic dissection (AD) rats. For this purpose a rat model of aortic dissection (AD) was constructed by giving drinking water containing 0.08% β-aminopropionitrile (BAPN) to rats, HE staining was used to observe the pathological changes of the aorta in AD rats; the diseased blood vessels of AD rats were taken for primary culture and passage of VSMCs, the morphology of VSMCs was observed, and VSMCs were identify with immunofluorescence staining; VSMCs were treated with culture media containing 0, 1000, 2000, 3000, 4000, 5000, 6000, 7000 U/mL ulinastatin, and MTT kit was used to determine the effect of ulinastatin on VSMC proliferation in AD rats; the VSMC of AD rats were divided into blank group (normal culture), ulinastatin group (medium containing 5000 U/mL ulinastatin), Sirt1 inhibitor group (medium containing 1 μmol/L EX527), ulinastatin + Sirt1 inhibitor group (medium containing 5000 U/mL ulinastatin, 1 μmol/L EX527), flow cytometry was used to detect the VSMC apoptosis in each group, WB was used to detect the expression of VSMC apoptosis-related proteins and Sirt1/FoxO3a pathway-related proteins in each group. Findings suggested that the aortic wall of AD rats was thickened, and the dissection false cavity appeared; VSMC mostly presented different shapes such as triangles and stars, the immunofluorescence staining results showed that α-SMA was arranged in the cytoplasm in the form of myofilaments, showing green fluorescence, and the nucleus showed blue fluorescence, and the rate of positive cells was more than 95%; various doses of ulinastatin had a certain inhibitory effect on the proliferation of VSMC, and 5000 U/mL ulinastatin had a higher proliferation inhibition rate; compared with the blank group, the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the ulinastatin group were significantly increased, and the Bcl-2 protein expression was significantly decreased (P<0.05), the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the Sirt1 inhibitor group were significantly decreased, and the Bcl-2 protein expression was significantly increased (P<0.05); compared with the ulinastatin group, the VSMC apoptosis rate, Caspase-3, Bax protein, Sirt1/FoxO3a pathway related protein expression in the ulinastatin + Sirt1 inhibitor group were significantly decreased, and the Bcl-2 protein expression was significantly increased (P<0.05). It was concluded that ulinastatin can inhibit the proliferation of VSMCs in AD rats and promote their apoptosis, which may be achieved by activating the Sirt1/FoxO3a pathway.

Rehabilitation facilitates functional improvement following intravenous infusion of mesenchymal stem cells in the chronic phase of cerebral ischemia in rats

The primary objective of this study was to investigate the potential facilitating effects of daily rehabilitation for chronic cerebral ischemia following the intravenous infusion of mesenchymal stem cells (MSC) in rats. The middle cerebral artery (MCA) was occluded by intraluminal occlusion using a microfilament (MCAO). Eight weeks after MCAO induction, the rats were used as a chronic cerebral ischemia model. Four experimental groups were studied: Vehicle group (medium only, no cells); Rehab group (vehicle + rehabilitation), MSC group (MSC only); and Combined group (MSC + rehabilitation). Rat MSCs were intravenously infused eight weeks after MCAO induction, and the rats received daily rehabilitation through treadmill exercise for 20 min. Behavioral testing, lesion volume assessment using magnetic resonance imaging (MRI), and histological analysis were performed during the observation period until 16 weeks after MCAO induction. All treated animals showed functional improvement compared with the Vehicle group; however, the therapeutic efficacy was greatest in the Combined group. The combination therapy is associated with enhanced neural plasticity shown with histological analysis and MRI diffusion tensor imaging. These findings provide behavioral evidence for enhanced recovery by combined therapy with rehabilitation and intravenous infusion of MSCs, and may form the basis for the development of clinical protocols in the future.

Effects of Acetaminophen on the Reproductive Parameters and the Ameliorative Effects of Rutin and Mesenchymal Stem Cells in Male Rats

The present study has been designed to evaluate the effects of acetaminophen on the reproductive parameters and the ameliorative effects of rutin and Mesenchymal stem cells in male rats. The rats were divided into two studies: firstly, twenty young male rats were used as a source of bone marrow-derived MSCs. Secondly, fifty adult male rats were divided into 5 groups: Control group (G1); rats were administrated of AAP (750 mg/kg b.w.) every 72h for 21 days, then left for 30 and 60 days without treatment (G2), rats were administrated of AAP for 21 days then treated with RT (25mg/kg b.w/d) for 30 and 60 days (G3), rats were administrated of AAP for 21 days then the rats were injected by BM-MSCs (1.5x 106 cells in 0.5 PBS) in the tail vein for 30 and 60 days (G4), and rats were administrated of AAP for 21 days then the rats were injected by BM-MSCs in the tail vein, then treated with RT for 30 and 60 days (G5). Administration of AAP produced a significant decrease in the mean value of the Te, LH, and FSH levels after 30 and 60 days as compared to the C groups. Whereas rats treated with RT and MSCs showed a significantly high increase in the mean values of these hormone levels after 30 and 60 days compared with the AAP group. In conclusion, this study demonstrated that RT and MSCs when treated in combination, are protected against the AAP-induced decrease in the hormone level of testicular.

Screening of potent RIPK3 inhibitors to attenuate necroptosis and inflammation in mouse traumatic brain injury models

Necroptosis is a type of cell death that occurs when cells are exposed to external stressors such as inflammation, infections, or injury. In necroptosis, cells use a different set of proteins including: receptor-interacting kinase 1 (RIPK1 or RIP1), receptor-interacting kinase 3 (RIPK3 or RIP3) and the phosphorylation of its substrate mixed lineage kinase domain-like protein (MLKL) and pathways to trigger their own death. Mutations in the gene encoding RIPK3 have been associated with many diseases, including neurodegenerative diseases, neuroinflammatory diseases, inflammatory diseases，tumors, and it is being studied as a potential target for inflammatory injury therapy. RIPK3 has also been implicated in the pathology of neuroinflammation following Traumatic brain injury and is currently being explored as a potential therapy. We screened through necroptosis blocking compounds, a library of FDA-approved compounds. We found four compounds:1D6-Foretinib GSK1363089; 15F6-Poziotinib (HM781-36B); 15F9-Dasatinib monohydrate; 15A10-Pexmetinib (ARRY-614); acts as potent inhibitors of necroptosis (Necroptosis Blocking Compounds, NBCs) by blocking the RIPK3 kinase activity. These four compounds effectively block necroptosis induced by death receptor ligands Toll-like receptors as well as viral infections in human, rat and mouse cells. The cellular activation of RIPK3 and MLKL stimulated by necroptosis was strongly inhibited by NBCs. The compounds are promising for targeting RIPK3 kinase activity, thereby preventing necroptosis and inflammatory responses. In our study, we explored the role of NBCs in neuroprotection after traumatic brain injury. It's effectiveness in traumatic brain injury animal models and favorable safety profiles make it a potential candidate for the advances of new therapies for necroptosis-associated neuroinflammatory disorders.

Protective role of eugenol against diabetes-induced oxidative stress, DNA damage, and apoptosis in rat testes.

Diabetes mellitus, a metabolic disorder alters gonadal development and spermatogenesis,reactive oxygenspecies production, DNA damage, and apoptosis, which subsequently lead to male subfertility. Eugenol is an antioxidant, traditionally used as medication for digestive disorders and antioxidant therapy, decrease transport of glucose from GIT to systemic circulation. This experiment was aimed to decipher cellular and molecular insights of eugenol in protecting diabetic germ cells in rats. Rats were assigned randomly into five groups: control, eugenol control (Eugenol 400; EUG), diabetic (DIA), diabetic + eugenol 100 (DIA + EUG 100), and diabetic + eugenol 400 (DIA + EUG 400). EUG 400 and DIA + EUG 400 groups received 400 mg/kg eugenol orally. DIA + EUG 100 group received 100 mg/kg eugenol. Treatment was conducted for 4 weeks. Type 1 diabetes was induced by injecting a single i.p. dose of streptozotocin (55 mg/kg). Morphometric, biochemical, sperm parameters, oxidative stress, hormonal levels, histopathology, and fibrosis in the testis and epididymis, were evaluated. DNA damage was evaluated using halo and comet assays; DNA fragmentation and apoptosis using TUNEL assay. Eugenol treatment significantly normalized biochemical parameters, reduced MDA while increased albumin and GSH levels in diabetes. Eugenol significantly increased sperm numbers, motility and attenuated abnormal sperm head morphology in diabetes. Moreover, eugenol significantly reversed diabetes-induced cellular damages, altered spermatogenesis, and collagen deposition in testis and epididymis. It also significantly attenuated diabetes-associated DNA breaks and apoptosis. These findings suggest that 4 weeks treatment with 400 mg/kg of eugenol could be beneficial for diabetic patients to prevent subfertility.

Ex Vivo Production of IL-1β and IL-10 by Activated Blood Cells of Wistar Rats with Different Resistance to Hypoxia after Systemic Inflammatory Response Syndrome.

We studied spontaneous and ex vivo activated cytokine production by blood cells of male Wistar rats with different resistance to hypoxia against the background of an LPS-induced systemic inflammatory response. In rats with low (LR) and high resistance (HR) to hypoxia, the number of leukocytes, granulocytes, and peripheral blood lymphocytes was determined, the levels of spontaneous and stimulated production of IL-1β and IL-10 and their ratio were assessed ex vivo. Against the background of a systemic inflammatory response, only HR animals showed a decrease in spontaneous and stimulated production of IL-1β and spontaneous production of IL-10. The IL-1β/IL-10 ratio decreased only in LR rats during the development of a systemic inflammatory response, while in HR animals, no changes in this indicator were observed. The obtained data suggest a high proinflammatory potential of blood cells in LR rats, which apparently determines the development of a more severe course of the systemic inflammatory response.