Repair effect of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles on ovarian injury induced by cisplatin.

Small extracellular vesicles (sEVs) secreted by human umbilical cord have therapeutic effects on various degenerative diseases. However, the characteristics and potential functions of human umbilical cord mesenchymal stem cells (huMSCs)-derived sEVs, especially the role of premature ovarian failure (POF), are poorly understood. Here, we isolated and characterized huMSCs and their sEVs. huMSCs highly expressed CD73, CD90, and CD105. huMSC-sEVs showed typical exosomal features, highly expressing CD9, TSG101, and CD63. It was shown that huMSC-sEVs could be taken up by granulosa cells (GCs) and damaged ovarian tissue, which increased the levels of hormone secretion and reduced GCs apoptosis. We further confirmed that the levels of follicle-stimulating hormone in rat serum decreased dramatically, while the levels of estrogen (E2）and anti-mullerian hormone (AMH) increased significantly with the treatment of huMSC-sEVs. Meanwhile, huMSC-sEVs treatment greatly reduced cell apoptosis and autophagy, while increased the phosphorylation levels of p-PI3K and p-Akt. Therefore, treatment with huMSC-sEVs significantly inhibited GCs apoptosis, improved ovarian morphology, promoted follicular development, inhibited follicular over-atresia, and improved ovarian reserve capacity in POF rats. Our study verified that activation of PI3K/Akt signaling pathway and regulation of cellular autophagy, thus reducing GCs death, are the mechanisms by which huMSC-sEVs restore ovarian tissue function.

Oxymatrine blocks the NLRP3 inflammasome pathway, partly downregulating the inflammatory responses of M1 macrophages differentiated from THP-1 monocytes.

Many chronic inflammatory diseases, such as autoimmune inflammation, are associated with M1 macrophages, and the key to their treatment is blocking inflammation. Oxymatrine (OMT), a traditional Chinese medicine, has a marked anti-inflammatory effect. However, its anti-inflammatory target and mechanism in M1 cells remain unclear, which limits its clinical application. In this study, we investigated the anti-inflammatory effects of oxymatrine (OMT) on the M1 inflammatory response. We also determined the relationship between OMT treatment and the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway with OMT treatment. To this end, we induced the differentiation of human peripheral blood monocytes (THP-1) into M1 cells. THP-1 cells were induced with a phorbol ester (phorbol-12-myristate-13-acetate (PMA)) and differentiated into naïve M0 macrophages. M0 cells were induced into M1 cells using lipopolysaccharide (LPS). The experimental groups were divided into the M0 macrophage group (NC), M1 inflammatory response group (LPS group), and M1 group treated with different concentrations of OMT (LPS + OMT-L, LPS + OMT-M, LPS + OMT-H). The cells in the OMT-treated groups were treated with OMT for 6 h, followed by LPS for 24 h, and the LPS group was treated with LPS only. The resulting supernatants and cells were collected. The secretion levels of NO were detected by the Griess method and the secretion levels of TNF-α and IL-1ß in the supernatants were detected by the ELISA method. The secretion levels of these inflammatory factors were reduced in every OMT-treated group compared to the LPS group (P < 0.01), and the most significant reductions were found in the OMT-H group (P < 0.0001). By western blotting, the protein expression levels of TLR4, NF-κB, NLRP3, and Caspase-1 were all found to be downregulated in the cells of OMT-treated groups compared to the LPS group (P < 0.0001). In situ changes in NLRP3 expression were observed using immunofluorescence. The fluorescence intensity of NLRP3 in M1 cells was weaker in all OMT intervention groups than in the LPS group (P < 0.001). In conclusion, OMT has significant anti-inflammatory effects on the M1 inflammatory responses, and the TLR4/NF-κB/NLRP3 pathway was blocked proportional to the concentration of OMT.

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate Parkinson's disease and neuronal damage through inhibition of microglia.

Microglia-mediated inflammatory responses have been shown to play a crucial role in Parkinson's disease. In addition, exosomes derived from mesenchymal stem cells have shown anti-inflammatory effects in the treatment of a variety of diseases. However, whether they can protect neurons in Parkinson's disease by inhibiting microglia-mediated inflammatory responses is not yet known. In this study, exosomes were isolated from human umbilical cord mesenchymal stem cells and injected into a 6-hydroxydopamine-induced rat model of Parkinson's disease. We found that the exosomes injected through the tail vein and lateral ventricle were absorbed by dopaminergic neurons and microglia on the affected side of the brain, where they repaired nigral-striatal dopamine system damage and inhibited microglial activation. Furthermore, in an in vitro cell model, pretreating lipopolysaccharide-stimulated BV2 cells with exosomes reduced interleukin-1ß and interleukin-18 secretion, prevented the adoption of pyroptosis-associated morphology by BV2 cells, and increased the survival rate of SH-SY5Y cells. Potential targets for treatment with human umbilical cord mesenchymal stem cells and exosomes were further identified by high-throughput microRNA sequencing and protein spectrum sequencing. Our findings suggest that human umbilical cord mesenchymal stem cells and exosomes are a potential treatment for Parkinson's disease, and that their neuroprotective effects may be mediated by inhibition of excessive microglial proliferation.

Aqueous extract of Salvia miltiorrhiza Bunge reduces blood pressure through inhibiting oxidative stress, inflammation and fibrosis of adventitia in primary hypertension.

Background: Hypertension is a major risk factor for cardiovascular diseases and the leading cause of mortality worldwide. Despite the availability of antihypertensive drugs, alternative treatments are needed due to the adverse events associated with their use. Previous studies have shown that SABP, a combination of aqueous active metabolites of Salvia Miltiorrhiza Bunge DSS, Sal-A, Sal-B and PAL, has a significant antihypertensive effect. However, the underlying mechanisms remain unknown. Objective: This study aimed to determine the effects of SABP on vascular inflammation, oxidative stress, and vascular remodeling in spontaneously hypertensive rats (SHRs). Additionally, the response of adventitial fibroblasts in SHRs to SABP treatment was also studied, including their proliferation, differentiation, and migration. Methods: SABP or perindopril (positive control) were administered intraperitoneally to SHRs, and systolic blood pressure was measured using a tail-cuff approach. The effects of SABP on oxidative stress, inflammation, and vascular remodeling were investigated by transmission electron microscopy, histochemical staining, and Western blot. Adventitial fibroblasts were isolated and cultured from the adventitia of thoracic aorta in SHR and WKY rats. CCK8 assay, wound healing method and immunostaining were used to observe the effect of SABP on fibroblasts proliferation, migration and transformation into myofibroblasts. Moreover, Western blot analysis was also performed to detect the proteins related to oxidative stress, inflammation and fibrosis in adventitial fibroblasts. Results: SHRs displayed higher blood pressure with significant vascular remodeling compared to WKY rats. The thoracic aorta and adventitial fibroblasts of SHRs exhibited significant oxidative stress, inflammation and fibrosis. SABP treatment repressed oxidative stress, inflammatory reaction and vascular remodeling of thoracic aorta in SHR through the ROS/TLR4/NF-κB signaling pathway, and inhibited fibrosis of thoracic aorta. Additionally, SABP inhibited the proliferation and migration of adventitial fibroblasts and their transformation to myofibroblasts in vitro through the TGFß/Smad3 signaling pathway. Conclusion: These findings suggest that SABP have potential as an alternative treatment for hypertension by ameliorating oxidative stress, inflammation and fibrosis. Further research is needed to fully understand the mechanisms underlying the effects of SABP.

Salvia miltiorrhiza Extract Prevents the Occurrence of Early Atherosclerosis in Apoe -/- Mice via TLR4/ NF-kB Pathway.

OBJECTIVE: Salvia miltiorrhiza (SM) contains four major aqueous active ingredients, which have been isolated, purified and identified as danshensu (DSS), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and protocatechuic aldehyde (PAL), A mixture of these four ingredients is called SABP. Although aqueous extract from Salvia miltiorrhiza has been traditionally used to treat cardiovascular diseases, the efficacy and function of the optimal ratio of SABP in preventing and treating cardiovascular diseases remain unknown. This study aims to explore the antiinflammatory mechanisms underlying the attenuation of atherosclerosis development by aqueous extract from Salvia miltiorrhiza. METHODS: Male ApoE-/- mice (6 weeks) were randomly allocated into three groups: the model group (Model), the SABP group (SABP), and the rosuvastatin calcium group (RC). Male C57BL/6 mice (6 weeks) were used as a control group. All mice were fed with an ordinary diet. After 8 weeks of treatment, the lipid profiles in serum and the lactate dehydrogenase (LDH) and creatine kinase (CK) in heart tissue were measured using an automatic biochemical analyzer. Alterations of the thoracic aorta and the heart were assessed using Hematoxylin and eosin staining. The protein expression of Toll-like receptor 4 (TLR4), TGF beta-activated kinase 1 (TAK1), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the heart tissue were determined though immunohistochemistry and western blotting analysis. RESULTS: The serum low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC) levels were increased, and the high-density lipoprotein cholesterol (HDL-C) level was decreased in ApoE-/- mice. SABP significantly decreased serum lipid levels and improved histopathology in the thoracic aorta. In addition. SABP treatment inhibited the expression of TLR4, TAK1, NF-κB, IL-6 and TNF-α in the heart in ApoE-/- mice. The LDH and CK in the heart did not differ significantly among different groups, and the heart did not have obvious pathological changes. CONCLUSION: These findings indicated that SABP may exert an anti-atherosclerotic effect by lowering blood lipids and inhibiting inflammatory response via TLR4/ NF-κB signaling pathway.

Expression of recombinant human Apolipoprotein A-IMilano in Nicotiana tabacum.

Apolipoprotein A-IMilano (Apo A-IMilano) is a natural mutant of Apolipoprotein. It is currently the only protein that can clear arterial wall thrombus deposits and promptly alleviate acute myocardial ischemia. Apo A-IMilano is considered as the most promising therapeutic protein for treating atherosclerotic diseases without obvious toxic or side effects. However, the current biopharmaceutical platforms are not efficient for developing Apo A-IMilano. The objectives of this research were to express Apo A-IMilano using the genetic transformation ability of N. tabacum. The method is to clone the coding sequence of Apo A-IMilano into the plant binary expression vector pCHF3 with a Flag/His6/GFP tag. The constructed plasmid was transformed into N. tabacum by a modified agrobacterium-mediated method, and transformants were selected under antibiotic stress. PCR, RT-qPCR, western blot and co-localization analysis was used to further verify the resistant N. tabacum. The stable expression and transient expression of N. tabacum were established, and the pure product of Apo A-IMilano was obtained through protein A/G agarose. The results showed that Apo A-IMilano was expressed in N. tabacum with a yield of 0.05 mg/g leaf weight and the purity was 90.58% ± 1.65. The obtained Apo A-IMilano protein was subjected to amino acid sequencing. Compared with the theoretical sequence of Apo A-IMilano, the amino acid coverage was 86%, it is also found that Cysteine replaces Arginine at position 173, which indicates that Apo A-IMilano, a mutant of Apo A-I, is accurately expressed in N. tabacum. The purified Apo A-IMilano protein had a lipid binding activity. The established genetic modification N. tabacum will provide a cost-effective system for the production of Apo A-IMilano. Regarding the rapid propagation of N. tabacum, this system provides the possibility of large-scale production and accelerated clinical translation of Apo A-IMilano.

An Optimal Combination of Chemically Pure Compounds from Salvia miltiorrhiza for Inhibiting Cell Proliferation.

OBJECTIVE: Salvia miltiorrhiza (SM) is a traditional Chinese medicine used clinically to treat cardiovascular diseases, including atherosclerosis and myocardial infarction. Its therapeutic effect has been confirmed by many clinical and pharmacological studies. However, the optimal formulation of active ingredients in SM for treating cardiovascular diseases remains unclear. In this study, we determined the ratio of the optimal compatibility of SM ingredients DSS, Sal-A, Sal-B, and PAL (SABP)with a uniform and orthogonal optimized experimental design. In addition, we determined the anti-oxidation effect of SABP using Adventitial Fibroblasts (AFs). METHODS: By using a combination of uniform and orthogonal designs, we determined the optimal formulation of aqueous extract from SM. MTT assay was used to determine the inhibitory effects of these 4 components of SM on the AFs, which were isolated and cultured from the aorta. The reactive oxygen species (ROS) production in AFs was compared before and after SABP treatment. RESULTS: The optimal formulation of these 4 aqueous extracts from SM were 150 : 7 : 300 : 500, and their concentrations were S(1.5×10-4 mol/L), A(7×10-6 mol/L), B(3×10-4 mol/L), and P(5×10-4 mol/L). There were some synergies between these 4 components. Moreover, SABP decreased ROS production in AFs. CONCLUSION: These findings suggest that SABP inhibits the proliferation and oxidation stress in AFs. The present study provides new evidence that the efficacy and function generated from the optimal formulation of active ingredients in SM are better than lyophilized powder of SM.

Human Umbilical Cord Mesenchymal Stem Cells Improve Locomotor Function in Parkinson's Disease Mouse Model Through Regulating Intestinal Microorganisms.

Parkinson's disease (PD) is a progressive neurological disorder characterized by loss of neurons that synthesize dopamine, and subsequent impaired movement. Umbilical cord mesenchymal stem cells (UC-MSCs) exerted neuroprotection effects in a rodent model of PD. However, the mechanism underlying UC-MSC-generated neuroprotection was not fully elucidated. In the present study, we found that intranasal administration of UC-MSCs significantly alleviated locomotor deficits and rescued dopaminergic neurons by inhibiting neuroinflammation in a PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, a toxic agent which selectively destroys nigrostriatal neurons but does not affect dopaminergic neurons elsewhere). Furthermore, UC-MSC treatment altered gut microbiota composition characterized by decreased phylum Proteobacteria, class Gammaproteobacteria, family Enterobacteriaceae, and genus Escherichia-Shigella. In addition, the neurotransmitter dopamine in the striatum and 5-hydroxytryptamine in the colon were also modulated by UC-MSCs. Meanwhile, UC-MSCs significantly maintained intestinal goblet cells, which secrete mucus as a mechanical barrier against pathogens. Furthermore, UC-MSCs alleviate the level of TNF-α and IL-6 as well as the conversion of NF-κB expression in the colon, indicating that inflammatory responses were blocked by UC-MSCs. PICRUSt showed that some pathways including bacterial invasion of epithelial cells, fluorobenzoate degradation, and pathogenic Escherichia coli infection were significantly reversed by UC-MSCs. These data suggest that the beneficial effects were detected following UC-MSC intranasal transplantation in MPTP-treated mice. There is a possible neuroprotective role of UC-MSCs in MPTP-induced PD mice by cross talk between the brain and gut.

Exosomes derived from mesenchymal stem cells repair a Parkinson's disease model by inducing autophagy.

Parkinson's disease (PD) is a progressively debilitating neurodegenerative condition that leads to motor and cognitive dysfunction. At present, clinical treatment can only improve symptoms, but cannot effectively protect dopaminergic neurons. Several reports have demonstrated that human umbilical cord mesenchymal stem cells (hucMSCs) afford neuroprotection, while their application is limited because of their uncontrollable differentiation and other reasons. Stem cells communicate with cells through secreted exosomes (Exos), the present study aimed to explore whether Exos secreted by hucMSCs could function instead of hucMSCs. hucMSCs were successfully isolated and characterized, and shown to contribute to 6-hydroxydopamine (6-OHDA)-stimulated SH-SY5Y cell proliferation; hucMSC-derived Exos were also involved in this process. The Exos were purified and identified, and then labeled with PKH 26, it was found that the Exos could be efficiently taken up by SH-SY5Y cells after 12 h of incubation. Pretreatment with Exos promoted 6-OHDA-stimulated SH-SY5Y cells to proliferate and inhibited apoptosis by inducing autophagy. Furthermore, Exos reached the substantia nigra through the blood-brain barrier (BBB) in vivo, relieved apomorphine-induced asymmetric rotation, reduced substantia nigra dopaminergic neuron loss and apoptosis, and upregulated the level of dopamine in the striatum. These results demonstrate that hucMSCs-Exos have a treatment capability for PD and can traverse the BBB, indicating their potential for the effective treatment of PD.

Active compounds of herbs ameliorate impaired cognition in APP/PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD), the most common cause of dementia, is a neurodegenerative disorder characterized by amyloid plaque accumulations, intracellular tangles and neuronal loss in certain brain regions. It has been shown that a disturbance of normal iron metabolism contributes to the pathophysiology of AD. However, the mechanism underlying abnormal iron load in the brain of AD patients is unclear. The frontal cortex, an important brain structure for executive function, is one of the regions affected by AD. We investigated the beneficial effects of active compounds of Epimedium, Astragaoside and Puerarin on iron metabolism in the frontal cortex of six-month-old APPswe/PS1ΔE9 (APP/PS1) double transgenic mouse, a model of AD. Treatment with the active compounds reduced cognitive and memory deficits and damaged cell ultrastructure in APP/PS1 mice. These beneficial effects were associated with changes in expression levels of iron metabolism proteins in the frontal cortex, including divalent metal transporter with iron response element (DMT1-with IRE), divalent metal transporter without iron response element (DMT1-without IRE), transferrin (TF) and transferring receptor 1 (TfR1); three release proteins including the exporter ferroportin 1 (Fpn1), ceruloplasmin (CP) and hephaestin (HEPH), one increased storage iron protein ferritin and one iron regulating hormone hepcidin. These findings suggest that the active compounds improve cognition and memory in brain neurodegenerative disorders and these beneficial effects are associated with reduced impairment of iron metabolism. This study may provide a new strategy for developing novel drugs to treat AD.

[Effects of angiotensin II and its receptor blockers on migration and endothelin-1 expression of rat vascular adventitial fibroblast subpopulations].

The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.

[Estrogen receptor subtypes and the regulatory effect of receptor ligand binding on gene transcription].

This review introduces the structure, functions, tissue distribution, physiologic roles of estrogen receptor subtypes (ER alpha and ER beta) along with transcriptional activities of estrogen receptor ligands and the mechanism of the modulatory pathway and tissue specific property of selective estrogen receptor modulators (SERMs), and phytoestrogens. This article is to provide a systemic approach for increasing the selectivity of estrogenic drug and optimizing the clinically based strategy of drug design. Differences exist between ER alpha and ER beta in structure, function, tissue distribution, physiologic roles and response to ligands, as well as the regulating effect on gene transcription, which are mainly determined by different co-regulators they recruit respectively.