Novel insights into the protective effects of leonurine against acute kidney injury: Inhibition of ER stress-associated ferroptosis via regulating ATF4/CHOP/ACSL4 pathway.

Acute kidney injury (AKI) is a common and serious global health problem with high risks of mortality and the development of chronic kidney diseases. Leonurine is a unique bioactive component from Leonurus japonicus Houtt. and exerts antioxidant, antiapoptotic or anti-inflammatory properties. This study aimed to explore the benefits of leonurine on AKI and the possible mechanisms involved, with a particular foc on the regulation of ferroptosis and endoplasmic reticulum (ER) stress. Our results showed that leonurine exhibited prominent protective effects against AKI, as evidenced by the amelioration of histopathological alterations and reduction of renal dysfunction. In addition, leonurine significantly suppressed ferroptosis in AKI both in vivo and in vitro by effectively restoring ultrastructural abnormalities in mitochondria, decreasing ASCL4 and 4-HNE levels, scavenging reactive oxygen species (ROS), as well as increasing GPX4 and GSH levels. In parallel, leonurine also markedly mitigated ER stress via down-regulating PERK, eIF-2α, ATF4, CHOP and CHAC1. Further studies suggested that ER stress was closely involved in erastin-induced ferroptosis, and leonurine protected tubular epithelial cells in vitro by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway. Mechanistically, ATF4 silencing in vitro regulated CHOP and ACSL4 expressions, ultimately weakening both ER stress and ferroptosis. Notably, analyses of single-cell RNA sequencing data revealed that ATF4, CHOP and ASCL4 in renal tubular cells were all abnormally upregulated in patients with AKI compared to healthy controls, suggesting their contributions to the pathogenesis of AKI. Altogether, these findings suggest that leonurine alleviates AKI by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway, thus providing novel mechanisms for AKI treatment.

[Effect and mechanism of Jiedu Huoxue Decoction in regulating YAP/ACSL4 pathway to inhibit ferroptosis in treatment of acute kidney injury].

Jiedu Huoxue Decoction(JDHX), first recorded in the Correction on Errors in Medical Works by WANG Qing-ren, is an effective formula screened out from ancient formulas by the traditional Chinese medicine(TCM) master ZHANG Qi to treat acute kidney injury(AKI) caused by heat, toxicity, stasis, and stagnation. This paper elucidated the therapeutic effect of JDHX on AKI and probed into the potential mechanism from ferroptosis. Thirty-two male C57BL/6 mice were randomized into four groups(n=8): normal, model, and low-and high-dose JDHX. Since the clinical treatment of AKI depends on supportive or alternative therapies and there is no specific drug, this study did not include a positive drug group. The low dose of JDHX corresponded to half of clinically equivalent dose, while the high dose corresponded to the clinically equivalent dose. Mice were administrated with JDHX by gavage daily for 7 consecutive days, while those in the normal group and the model group were administered with the corresponding volume of distilled water. On day 5 of drug administration, mice in other groups except the normal group were injected intraperitoneally with cisplatin solution at a dose of 20 mg·kg~(-1) to induce AKI, and the normal group was injected with saline. All of the mice were sacrificed 72 h after modeling, blood and kidney samples were collected for subsequent analysis. The levels of serum creatine(Scr) and blood urea nitrogen(BUN) were measured by the commercial kits. The expression level of kidney injury molecule 1(KIM-1) in the serum was measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin(HE) staining, periodic acid-Schiff(PAS) staining, and Prussian blue staining were employed to observe the pathological changes, glycogen deposition, and iron deposition, respectively, in the renal tissue. In addition, the levels of glutathione(GSH), superoxide dismutase(SOD), and catalase(CAT) in the renal tissue were examined by biochemical colorimetry. Western blot was performed to determine the protein levels of acyl-CoA synthetase long chain family member 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), and Yes-associated protein(YAP, a key molecule in the Hippo pathway) in the renal tissue. Immunohistochemistry was then employed to detect the location and expression of YAP in the renal tissue. Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was performed to measure the mRNA levels of ACSL4 and glutathione peroxidase 4(GPX4). Compared with the normal group, the model group showed elevated serum levels of Scr, BUN, and KIM-1. In the AKI model group, the tubular epithelial cells underwent atrophy and necrotic detachment, disappearance of brush border, and some tubules became protein tubules or experienced vacuole-like degeneration. In addition, this group presented widening of the interstitium or even edema, increased renal tubule injury score, and obvious glycogen and iron deposition in parts of the renal tissue. Moreover, the model group had lower GSH, SOD, and CAT levels, higher ASCL4 and LPCAT3 levels, and lower GPX4 expression and higher YAP expression than the normal group. Compared with the model group, high dose of JDHX effectively protected renal function, lowered the levels of Scr, BUN and KIM-1, alleviated renal pathological injury, reduced glycogen and iron deposition, and elevated the GSH, SOD, and CAT levels in the renal tissue. Furthermore, JDHX down-regulated the protein levels of ACSL4, LPCAT3, and YAP and up-regulated the level of GPX4, compared with the model group. In conclusion, JDHX can protect mice from cisplatin-induced AKI by inhibiting ferroptosis via regulating the YAP/ACSL4 signaling pathway.

3D Printing of Cobalt-Incorporated Chloroapatite Bioceramic Composite Scaffolds with Antioxidative Activity for Enhanced Osteochondral Regeneration.

Osteochondral defects are often accompanied by excessive reactive oxygen species (ROS) caused by osteoarthritis or acute surgical inflammation. An inflammatory environment containing excess ROS will not only hinder tissue regeneration but also impact the quality of newly formed tissues. Therefore, there is an urgent need to develop scaffolds with both ROS scavenging and osteochondral repair functions to promote and protect osteochondral tissue regeneration. In this work, by using 3D printing technology, a composite scaffold based on cobalt-incorporated chloroapatite (Co-ClAP) bioceramics, which possesses ROS-scavenging activity and can support cell proliferation, adhesion, and differentiation, is developed. Benefiting from the catalytic activity of Co-ClAP bioceramics, the composite scaffold can protect cells from oxidative damage under ROS-excessive conditions, support their directional differentiation, and simultaneously mediate an anti-inflammatory microenvironment. In addition, it is also confirmed by using rabbit osteochondral defect model that the Co-ClAP/poly(lactic-co-glycolic acid) scaffold can effectively promote the integrated regeneration of cartilage and subchondral bone, exhibiting an ideal repair effect in vivo. This study provides a promising strategy for the treatment of defects with excess ROS and inflammatory microenvironments.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

[Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway].

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 µmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 µmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 µmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 µmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 µmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 µmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Identification of a novel small-molecule inhibitor targeting TIM-3 for cancer immunotherapy.

PD-1/PD-L1 blockade has achieved substantial clinical results in cancer treatment. However, the expression of other immune checkpoints leads to resistance and hinders the efficacy of PD-1/PD-L1 blockade. T cell immunoglobulin and mucin domain 3 (TIM-3), a non-redundant immune checkpoint, synergizes with PD-1 to mediate T cell dysfunction in tumor microenvironment. Development of small molecules targeting TIM-3 is a promising strategy for cancer immunotherapy. Here, to identify small molecule inhibitors targeting TIM-3, the docking pocket in TIM-3 was analyzed by Molecular Operating Environment (MOE) and the Chemdiv compound database was screened. The small molecule SMI402 could bind to TIM-3 with high affinity and prevent the ligation of PtdSer, HMGB1, and CEACAM1. SMI402 reinvigorated T cell function in vitro. In the MC38-bearing mouse model, SMI402 inhibited tumor growth by increasing CD8+ T and natural killing (NK) cells infiltration at the tumor site, as well as restoring the function of CD8+ T and NK cells. In conclusions, the small molecule SMI402 shows promise as a leading compound which targets TIM-3 for cancer immunotherapy.

Metal-Organic Framework-Based Nanoagents for Effective Tumor Therapy by Dual Dynamics-Amplified Oxidative Stress.

Overproduction of reactive oxygen species (ROS) within tumors can cause oxidative stress on tumor cells to induce death, which has motivated us to develop ROS-mediated tumor therapies, such as typical photodynamic therapy (PDT) and Fenton reaction-mediated chemodynamic therapy (CDT). However, these therapeutic modalities suffer from compromised treatment efficacy owing to their limited generation of highly reactive ROS in a tumor microenvironment (TME). In this work, a nanoscale iron-based metal-organic framework, MIL-101(Fe), is synthesized as a Fenton nanocatalyst to perform the catalytic conversion of hydroxyl radicals (·OH) from hydrogen peroxide (H2O2) under the acidic environment and as a biocompatible and biodegradable nanocarrier to deliver a 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) photosensitizer for light-activated singlet oxygen (1O2) generation. By coupling such chemodynamic/photodynamic effects, the photosensitizer-integrated nanoagents (MIL-101(Fe)@TCPP) could enable more ROS production within tumors to induce amplified oxidative damage for tumor-specific synergistic therapy. In vitro results show that MIL-101(Fe)@TCPP nanoagents achieve the acid-responsive CDT and effective PDT, and synergistic CDT/PDT provides an enhanced therapeutic effect. Ultimately, based on such synergistic therapy, MIL-101(Fe)@TCPP nanoagents cause a significant tumor growth inhibition in vivo without severe side effects, showing great potential for anti-tumor application.

Metallic Oxide-Induced Self-Assembly of Block Copolymers to Form Polymeric Hybrid Micelles with Tunable Stability for Tumor Microenvironment-Responsive Drug Delivery.

Since block copolymers are able to self-assemble into various polymeric architectures, it is intriguing to explore a unique self-assembly strategy for polymers. Two different metallic oxides [manganese dioxide (MnO2) and zinc oxide (ZnO)] are displayed herein to demonstrate this self-assembly mechanism of polymers. In situ generation of metallic oxides induces self-assembly of block copolymers to form polymeric hybrid micelles with tunable stability in aqueous solutions. These final ZnO-cross-linked polymeric micelles exhibited a high drug loading capacity of 0.41 mg mg-1 toward doxorubicin (DOX), whereas DOX-loaded ZnO-cross-linked polymeric micelles could be broken down into Zn2+ and polymer scraps, which facilitated drug release in tumor microenvironments. Both in vitro and in vivo investigations showed that the drug-loaded ZnO-cross-linked polymeric micelles effectively suppressed tumor growth. Accordingly, the present study demonstrates a novel strategy of polymer self-assembly for fabricating polymeric architectures that can potentially provide insight for developing other polymeric architectures.

Computer-aided design of PVR mutants with enhanced binding affinity to TIGIT.

BACKGROUND: TIGIT, as a novel immune checkpoint molecule involved in T cell and NK cell anergy, could induce the immune tolerance and escape through binding with its ligand PVR. Blockade of TIGIT/PVR is considered as a promising strategy in cancer immunotherapy. However, to facilitate the design of inhibitors targeting TIGIT/PVR, the structural characteristics and binding mechanism still need to be further studied. METHODS: In this study, molecular dynamics (MD) simulations and in silico mutagenesis were used to analyze the interaction between TIGIT and its ligand PVR. Then, PVR mutants were designed and their activities were determined by using TIGIT overexpressed Jurkat cells. RESULTS: The results suggested that the loops of PVR (CC' loop, C'C″ loop, and FG loop) underwent a large intra-molecular rearrangement, and more hydrogen bond crosslinking between PVR and TIGIT were formed during MD simulations. The potential residues for PVR to interact with TIGIT were identified and utilized to predict high affinity PVR mutants. Through the biological activity evaluation, four PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) with enhanced affinity to TIGIT were discovered, which could elicit more potent inhibitory effects compared with the wild type PVR. CONCLUSIONS: The MD simulations analysis provided new insights into the TIGIT/PVR interaction model, and the identified PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) could serve as new candidates for immunotherapy to block TIGIT/PVR. Video Abstract.

Sanqi Oral Solution Ameliorates Renal Ischemia/Reperfusion Injury via Reducing Apoptosis and Enhancing Autophagy: Involvement of ERK/mTOR Pathways.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI) is a significant health problem with high morbidity and mortality, yet prophylaxis strategies and effective drugs are limited. Sanqi oral solution (SQ) is a formulated medicine widely used in clinical settings to treat various renal diseases via enriching qi and activating blood circulation while its role on I/R-AKI remains unclear. Herein, by establishing rat I/R-AKI models, we intended to investigate the effect of SQ on the prevention of I/R-AKI and explore its underlying mechanisms. We demonstrated that SQ treatment significantly attenuated renal dysfunction of I/R-AKI, alleviated histological damages, inhibited renal apoptosis, and enhanced autophagy. Further investigation proved that SQ could significantly inhibit the activation of ERK and mTOR signaling pathways. Moreover, its renoprotective effect can be abolished by autophagy inhibitor 3-methyladenine (3-MA). Collectively, our results suggest that SQ exerts renoprotective effects on renal I/R injury via reducing apoptosis and enhancing autophagy, which are associated with regulating ERK/mTOR pathways.

[The Relationship between the Status of Human Papillomavirus 16/18 Infection and the Expression of Bcl-2 and Bax in Squamous Cell Carcinomas of the Lung.].

BACKGROUND: The relationship between the virus and carcinomas has attracted attention and China may be highly infected-District with HPV. It was unclear that whether there has been HPV infection in squamous cell carcinomas and the occurrence and development of lung cancer has the relation with HPV. This experiment applied situ hybridization and immunohistochemical techniques. This study is to investigate the relationship between the status of HPV16/18 infection between the expression of Bcl-2 and Bax in squamous cell carcinomas of the lung. METHODS: In 44 patients with squamous cell carcinomas of the lung, HPV 16/18 DNA was examined by in situ hybridization, expression of Bcl-2 and Bax was revealed by immunohistochemistry and koilocytes was detected in carcinomas tissues by morphologic examination. RESULTS: Of 44 patients with squamous cell carcinomas of the lung, 12 (27.27%) were found to be HPV negative. Twenty-three (52.27%) were found to be integrated form HPV, 9 (20.45%) were found to be large number of episomal form and a few integrated form HPV. And no simplex episomal form HPV was found. HPV 16/18 DNA could not be detected in 15 non-carcinomas tissues. A significantly higher 16/18 DNA positive rates in carcinomas tissues compared to non-carcinomas tissues (P<0.001). Of 44 patients, 20 (45.5%) were found to be Bcl-2 positive. There were significantly difference between integrated and other form HPV 16/18 in expression of Bcl-2 (P<0.05). CONCLUSIONS: HPV infection might be a particular agent in patient with squamous cell carcinomas of the lung. The levels of expression of Bcl-2 were significant higher in HPV positive patients than patients with HPV negative or episomal form HPV in squamous cell carcinomas of the lung.

Expression and purification of invasion plasmid antigen C of Shigella flexneri.

OBJECTIVE: To induce the expression of and purify invasion plasmid antigen C (IpaC) of Shigella flexneri for studying the pathogenesis of Shigella flexneri. METHODS: Prokaryotic expression plasmid pET32a-ipaC was constructed and incorporated into E.coli BL21 (lambda DE3). The engineered bacteria were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) to express IpaC, which was identified by SDS-PAGE and purified by QIA expressionist system. RESULTS: SDS-PAGE presented a band for the fusion protein with the relative molecular mass of approximately 63 000, whose expression reached up to 11% of the total protein of E.coli BL21(lambda DE3). After proper purification, a purity of the target fusion protein of over 90% was achieved when the concentration of imidazole for elution was 350 mmol/L. CONCLUSION: The recombinant plasmid pET32a-ipaC has been stably and efficiently expressed in E.coli BL21 (lambda DE3), and QIA expressionist purification system proves to be simple and highly efficient.