Ginsenoside compound K induces ferroptosis via the FOXO pathway in liver cancer cells.

Liver cancer is a common malignant tumor worldwide, traditional Chinese medicine is one of the treatment measures for liver cancer because of its good anti-tumor effects and fewer toxic side effects. Ginsenoside CK (CK) is an active component of ginseng. This study explored the mechanism by which CK induced ferroptosis in liver cancer cells. We found that CK inhibited the proliferation of HepG2 and SK-Hep-1 cells, induced ferroptosis of cells. Ferrostatin-1, an ferroptosis inhibitor, was used to verify the role of CK in inducing ferroptosis of liver cancer cells. Network pharmacological analysis identified the FOXO pathway as a potential mechanism of CK, and western blot showed that CK inhibited p-FOXO1. In cells treated with the FOXO1 inhibitor AS1842856, further verify the involvement of the FOXO pathway in regulating CK-induced ferroptosis in HepG2 and SK-Hep-1 cells. A HepG2 cell-transplanted tumor model was established in nude mice, and CK inhibited the growth of transplanted tumors in nude mice, p-FOXO1 was decreased in tumor tissues, and SLC7A11 and GPX4 expressions were also down-regulated after CK treatment. These findings suggested that CK induces ferroptosis in liver cancer cells by inhibiting FOXO1 phosphorylation and activating the FOXO signaling pathway, thus playing an antitumor role.

Intravitreal injection of new adeno-associated viral vector: Enhancing retinoschisin 1 gene transduction in a mouse model of X-linked retinoschisis.

Adeno-associated virus (AAV) vectors have been widely used in therapy to treat hereditary retinal diseases. But its transduction efficiency by intravitreal injection still needs to be improved. In this study, we investigated the transduction efficiency of AAV-DJ (K137R)-GFP in different retinal cells of normal mice, as well as the therapy effection of AAV-DJ (K137R)-Rs1 on retinal function and structure in Rs1-KO mice. The intravitreal injection of AAV-DJ (K137R)-GFP demonstrated that this vector transduced cells in all layers of the retina, including the inner nuclear layer and photoreceptor layer. The intravitreal injection of AAV-DJ (K137R)-Rs1 found that 3 months post-injection of this vector improved retinal function and structure in Rs1-KO mice. Our conclusion is that AAV-DJ (K137R) vector can efficiently and safely penetrate the inner limiting membrane and transduce different layers of retinal cells in the long term, as well as being able to continuously and efficiently express target therapeutic proteins, making it a candidate therapeutic vector for X-linked retinoschisis (XLRS).

De Novo Mutations Contributes Approximately 7% of Pathogenicity in Inherited Eye Diseases.

Purpose: The purpose of this study was to describe genotype-phenotype associations and novel insights into genetic characteristics in a trio-based cohort of inherited eye diseases (IEDs). Methods: To determine the etiological role of de novo mutations (DNMs) and genetic profile in IEDs, we retrospectively reviewed a large cohort of proband-parent trios of Chinese origin. The patients underwent a detailed examination and was clinically diagnosed by an ophthalmologist. Panel-based targeted exome sequencing was performed on DNA extracted from blood samples, containing coding regions of 792 IED-causative genes and their flanking exons. All participants underwent genetic testing. Results: All proband-parent trios were divided into 22 subgroups, the overall diagnostic yield was 48.67% (605/1243), ranging from 4% to 94.44% for each of the subgroups. A total of 108 IED-causative genes were identified, with the top 24 genes explaining 67% of the 605 genetically solved trios. The genetic etiology of 6.76% (84/1243) of the trio was attributed to disease-causative DNMs, and the top 3 subgroups with the highest incidence of DNM were aniridia (n = 40%), Marfan syndrome/ectopia lentis (n = 38.78%), and retinoblastoma (n = 37.04%). The top 10 genes have a diagnostic yield of DNM greater than 3.5% in their subgroups, including PAX6 (40.00%), FBN1 (38.78%), RB1 (37.04%), CRX (10.34%), CHM (9.09%), WFS1 (8.00%), RP1L1 (5.88%), RS1 (5.26%), PCDH15 (4.00%), and ABCA4 (3.51%). Additionally, the incidence of DNM in offspring showed a trend of correlation with paternal age at reproduction, but not statistically significant with paternal (P = 0.154) and maternal (P = 0.959) age at reproduction. Conclusions: Trios-based genetic analysis has high accuracy and validity. Our study helps to quantify the burden of the full spectrum IED caused by each gene, offers novel potential for elucidating etiology, and plays a crucial role in genetic counseling and patient management.

Study of the biological characteristics of human umbilical cord mesenchymal stem cells after long-time cryopreservation.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have considerable potential in cell therapy. Cryopreservation represents the gold standard in cell storage, but its effect on hUC-MSCs is still not well understood. The aim of this study was to investigate the effect of one year of cryopreservation and thawing on the biological characteristics of hUC-MSCs from the same donors. Fresh hUC-MSCs were cryopreserved in commercial freezing medium (serum-free CellBanker 2) at passage 2. After one year of cryopreservation, the hUC-MSCs were thawed and subcultured to passage 4. The comparison was performed in terms of followings: cell count, viability, morphology, proliferation capacity, differentiation potential and chromosomal stability. The total cell count and viability of hUC-MSCs before and after one year of cryopreservation were 1 × 107 and 96.34% and 0.943 × 107 and 93.81%, respectively. Cryopreserved and fresh hUC-MSCs displayed a similar cell doubling times, expressed the markers CD73, CD90, CD105 and were negative for the markers CD34, CD45, and HLA-DR. Karyotypes were found to be normal after one year of cryopreservation. The trilineage differentiation properties were maintained after cryopreservation. However, when compared to freshly isolated hUC-MSCs from the same donor, cryopreserved hUC-MSCs exhibited decreased expression of osteogenesis- and chondrogenesis-related genes including Runx2, Sox9, and Col1a1, and increased expression of adipogenesis-related genes. These results demonstrated that cryopreservation did not affect cell morphology, surface marker expression, cell viability, proliferative capacity, or chromosomal stability. However, the osteogenic and chondrogenic differentiation capacities of cryopreserved hUC-MSCs were slightly reduced compared with those of fresh cells from the same donor.

Intravitreal Injection of Human Retinal Progenitor Cells for Treatment of Retinal Degeneration.

BACKGROUND Retinal degeneration causes irreversible blindness. Human retinal progenitor cells (hRPCs) have the potential to treat retinal diseases. The vitreous cavity is a relatively immune-privileged site that is suitable for stem cell transplantation in the treatment of retinal diseases. This study aimed to evaluate the therapeutic efficacy and safety of intravitreal injection of hRPCs in retinal degeneration therapy. MATERIAL AND METHODS hRPCs were primary-cultured and injected into the vitreous cavity of RCS rats. To determine whether hRPCs formed teratomas in immune-deficient mice, hRPCs at different passages were transplanted into BALB/c-nu mice. The visual function was detected by electroretinography recording. Changes in the outer nuclear layer (ONL) were analyzed by histological testing and cell counting. The protective mechanism was further assessed by cytokine antibody array. RESULTS Intravitreal transplantation of hRPCs maintained retinal function and preserved retinal morphology. Importantly, grafted cells in the vitreous cavity were well tolerated, with no adverse effects. Teratoma was not formed in BALB/c-nu mice after hRPCs transplantation. The number of hRPCs-injected eyes and thickness of ONL in the hRPCs-treated group were higher than those in the untreated group and HBSS injection group. The cytokine antibody array revealed that hRPCs expressed GDF-15, PDGF-AA, EGF, and NT-4. CONCLUSIONS Our findings show that intravitreal injection of hRPCs is effective and safe in protecting photoreceptor cells in RCS rats, but were no longer effective at 12 weeks after transplantation. Moreover, hRPCs released multiple neurotrophic factors that may be involved in treating retinal disease.

The condition medium of mesenchymal stem cells promotes proliferation, adhesion and neuronal differentiation of retinal progenitor cells.

Retinal progenitor cell is a promising candidate in the treatment of retinal pigmentosa diseases. The limiting factors of stem cell transplantation are the proliferation and differentiation capacities of hRPCs, which may be governed by culture conditions. Previous studies have proved that the secretome of human Umbilical Cord Mesenchymal stem cells (hUCMSCs) and human Adipose derived stem cells (hADSCs), including more active cytokines and neurotrophic factors, have the paracrine potential of enhancing proliferation and differentiation in several cell types. The aim of this study was to investigate whether hRPCs could effectively proliferate, adhere and differentiate towards specific retinal cell types by treating with the condition medium (CM) of hUCMSCs (hUCMSCCM) or hADSCs (hADSCCM). Here, we show that hUCMSCCM or hADSCCM enhances the proliferation rate of the S and G2 phase cells, with an upregulation of Ki67 expression. Moreover, the upregulation expression of NF, Recoverin and Rhodopsin indicates that specialized retinal cells including ganglion cells and photoreceptors are favored over hRPCs differentiation due to hUCMSCCM or hADSCCM. Under FBS induced differentiation conditions, hRPCs treated with hUCMSCCM or hADSCCM increase the expression of retinal neuron and photoreceptor specific markers. These results suggest that hUCMSCCM and hADSCCM can stimulate the hRPC proliferation, promote its adherence and support hRPC neuronal and photoreceptor differentiation. These findings may provide a new strategy to improve the viability of hRPCs and photoreceptor differentiation capacities.