HIF-1α siRNA Research Articles

Development of a sequential release nanomaterial for co-delivery of hypoxia-induced tirapazamine and HIF-1α siRNA in cancer therapy

Unlike traditional drug carriers, sequential drug delivery systems can release different drugs in order, with the first released drug providing a prerequisite for the later released drug to maximize its function, thereby achieving stronger anti-tumor effects. Herein we constructed a temporal sequential system designated TPZ@MSN/HIF-1α siRNA@PDA@GOx (MTRPG) in which mesoporous silica nanoparticles were used as cores to load hypoxia induced chemotherapy drug tirapazamine (TPZ) and gene targeted nucleic acid drug HIF-1α siRNA, polydopamine (PDA) as acid –responsive coating as well as to realize photothermal therapy, and glucose oxidase (GOx) as the outermost layer to achieve starvation therapy and construct a deepened hypoxia to activate TPZ. Through in vitro and in vivo experiments, we demonstrated that the first released glucose oxidase catalyzed the oxidation of glucose, achieving starvation treatment while reducing the acidic environment and further exacerbating hypoxia in tumor cells. The reduced acidic conditions enabled the degradation of PDA, resulting in the release of loaded HIF-1α siRNA and TPZ. At the same time, PDA could also exert photothermal therapy under 808 nm near-infrared (808 nm NIR) laser irradiation. The later released hypoxia induced chemotherapy drug TPZ amplifies its anti-tumor activity under intensified hypoxia conditions. Meanwhile, the released HIF-1α siRNA interfered with the up-regulated HIF-1α induced by the deepened hypoxia condition, which caused hypoxia tolerance in tumors, reduced its expression activity, and achieved synergistic killing of tumor cells with chemotherapy. This work provides an effective multimodal synergistic therapy strategy to promote tumor therapeutic index, which may possess a promising future in clinical application.

Targeting hypoxia-inducible factor 1 alpha augments synergistic effects of chemo/immunotherapy via modulating tumor microenvironment in a breast cancer mouse model

Introduction: The immunosuppressive context of the tumor microenvironment (TME) is a significant hurdle in breast cancer (BC) treatment. Combinational therapies targeting cancer core signaling pathways involved in the induction of TME immunosuppressive milieu have emerged as a potent strategy to overcome immunosuppression in TME and enhance patient therapeutic outcomes. This study presents compelling evidence that targeting hypoxia-inducible-factor-1 alpha (Hif-1α) alongside chemotherapy and immune-inducing factors leads to substantial anticancer effects through modulation of TME. Methods: Chitosan (Cs)/Hif-1alpha siRNA nano-complex was synthesized by siRNA adsorption methods. Nanoparticles were fully characterized using dynamic light scattering and scanning electron microscope. Cs/Hif-1α siRNA cytotoxicity was measured by MTT assay. The anticancer effects of the combinational therapy were assessed in BALB/c bearing 4T1 tumors. qPCR and western blotting were applied to assess the expression of some key genes and proteins involved in the induction of immunosuppression in TME. Results: Hif-1α siRNA was successfully loaded in chitosan nanoparticles. Hif-1α siRNA nanocomplexes significantly inhibited the expression of Hif-1α. Triple combination therapy (Paclitaxel (Ptx) + Imiquimod (Imq) + Cs/Hif-1α siRNA) inhibited tumor growth and downregulated cancer progression genes while upregulating cellular-immune-related cytokines. Mice without Cs/Hif-1α siRNA treatments revealed fewer cancer inhibitory effects and more TME immunosuppressive factors. These results suggest that the inhibition of Hif-1α effects synergize with Ptx and Imq to inhibit cancer progression more significantly than other combinational treatments. Conclusion: Combining Hif-1α siRNA with Ptx and Imq is promising as a multimodality treatment. It has the potential to attenuate TME inhibitory effects and significantly enhance the immune system's ability to combat tumor cell growth, offering an inspiration of hope in the fight against BC.

A drug delivery system of HIF-1α siRNA nanoparticles loaded by mesenchymal stem cells on choroidal neovascularization.

Aim: To assess mesenchymal stem cells (MSCs) as carriers for HIF-1αsiRNA-loaded nanoparticles (NPs) for targeted therapy of experimental choroidal neovascularization (CNV).Materials & methods: A poly (lactic-co-glycolic acid) (PLGA)-core/lipid-shell hybrid NP was designed. The transfection efficacy of MSCs with the hybrid NPs was assessed. Mice were intravenously injected with MSCs after laser photocoagulation and CNV was assessed at 7days post-injection.Results & conclusion: The transfection efficiency of hybrid NPs into MSCs was 72.7%. HIF-1α mRNA expression in 661w cells co-cultured with MSC-hybrid-siRNA NPs was significantly lower. Intravenous delivery of MSC-hybrid-siRNA NPs greatly reduced CNV area and length. Intravenous injection of MSC-hybrid-siRNA NPs achieved therapeutic efficacy in reducing CNV area. The MSC-mediated homing enabled targeted inhibition of ocular angiogenesis.

Activating UCHL1 through the CRISPR activation system promotes cartilage differentiation mediated by HIF-1α/SOX9.

Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.

Endothelin-1 increases Na+-K+-2Cl- cotransporter-1 expression in cultured astrocytes and in traumatic brain injury model: An involvement of HIF1α activation.

Na+-K+-2Cl- cotransporter-1 (NKCC1) is present in brain cells, including astrocytes. The expression of astrocytic NKCC1 increases in the acute phase of traumatic brain injury (TBI), which induces brain edema. Endothelin-1 (ET-1) is a factor that induces brain edema and regulates the expression of several pathology-related genes in astrocytes. In the present study, we investigated the effect of ET-1 on NKCC1 expression in astrocytes. ET-1 (100 nM)-treated cultured astrocytes showed increased NKCC1 mRNA and protein levels. The effect of ET-1 on NKCC1 expression in cultured astrocytes was reduced by BQ788 (1 μM), an ETB antagonist, but not by FR139317 (1 μM), an ETA antagonist. The involvement of ET-1 in NKCC1 expression in TBI was examined using a fluid percussion injury (FPI) mouse model that replicates the pathology of TBI with high reproducibility. Administration of BQ788 (15 nmol/day) decreased FPI-induced expressions of NKCC1 mRNA and protein, accompanied with a reduction of astrocytic activation. FPI-induced brain edema was attenuated by BQ788 and NKCC1 inhibitors (azosemide and bumetanide). ET-1-treated cultured astrocytes showed increased mRNA and protein expression of hypoxia-inducible factor-1α (HIF1α). Immunohistochemical observations of mouse cerebrum after FPI showed co-localization of HIF1α with GFAP-positive astrocytes. Increased HIF1α expression in the TBI model was reversed by BQ788. FM19G11 (an HIF inhibitor, 1 μM) and HIF1α siRNA suppressed ET-induced increase in NKCC1 expression in cultured astrocytes. These results indicate that ET-1 increases NKCC1 expression in astrocytes through the activation of HIF1α.

Effect of apigetrin in pseudo-SARS-CoV-2-induced inflammatory and pulmonary fibrosis in vitro model

SARS-CoV-2 has become a global public health problem. Acute respiratory distress syndrome (ARDS) is the leading cause of death due to the SARS-CoV-2 infection. Pulmonary fibrosis (PF) is a severe and frequently reported COVID-19 sequela. In this study, an in vitro model of ARDS and PF caused by SARS-CoV-2 was established in MH-S, THP-1, and MRC-5 cells using pseudo-SARS-CoV-2 (PSCV). Expression of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and HIF-1α was increased in PSCV-infected MH-S and THP-1 cells, ARDS model, consistent with other profiling data in SARS-CoV-2-infected patients have been reported. Hypoxia-inducible factor-1 alpha (HIF-1α) siRNA and cobalt chloride were tested using this in vitro model. HIF-1α knockdown reduces inflammation caused by PSCV infection in MH-S and THP-1 cells and lowers elevated levels of CTGF, COLA1, and α-SMA in MRC-5 cells exposed to CPMSCV. Furthermore, apigetrin, a glycoside bioactive dietary flavonoid derived from several plants, including Crataegus pinnatifida, which is reported to be a HIF-1α inhibitor, was tested in this in vitro model. Apigetrin significantly reduced the increased inflammatory cytokine (IL-6, IL-1β, and TNF-α) expression and secretion by PSCV in MH-S and THP-1 cells. Apigetrin inhibited the binding of the SARS-CoV-2 spike protein RBD to the ACE2 protein. An in vitro model of PF induced by SARS-CoV-2 was produced using a conditioned medium of THP-1 and MH-S cells that were PSCV-infected (CMPSCV) into MRC-5 cells. In a PF model, CMPSCV treatment of THP-1 and MH-S cells increased cell growth, migration, and collagen synthesis in MRC-5 cells. In contrast, apigetrin suppressed the increase in cell growth, migration, and collagen synthesis induced by CMPSCV in THP-1 and MH-S MRC-5 cells. Also, compared to control, fibrosis-related proteins (CTGF, COLA1, α-SMA, and HIF-1α) levels were over two-fold higher in CMPSV-treated MRC-5 cells. Apigetrin decreased protein levels in CMPSCV-treated MRC-5 cells. Thus, our data suggest that hypoxia-inducible factor-1 alpha (HIF-1α) might be a novel target for SARS-CoV-2 sequela therapies and apigetrin, representative of HIF-1alpha inhibitor, exerts anti-inflammatory and PF effects in PSCV-treated MH-S, THP-1, and CMPVSC-treated MRC-5 cells. These findings indicate that HIF-1α inhibition and apigetrin would have a potential value in controlling SARS-CoV-2-related diseases.

Hypoxia-induced TRPM7 promotes glycolytic metabolism and progression in hepatocellular carcinoma

BackgroundHypoxia disrupts glucose metabolism in hepatocellular carcinoma (HCC). Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) plays an ontogenetic role. Thus, we aimed to explore the regulation of TRPM7 by hypoxia-induced factor (HIF) and its underlying mechanisms in HCC. Methodshypoxia was induced in multiple HCC cells using 1% O2 or CoCl2 treatment, and subsequently blocked using siRNAs targeting HIF-1α or HIF-2α as well as a HIF-1α protein synthesis inhibitor. The levels of HIF-1α and TRPM7 were assessed using quantitative PCR (qPCR) and Western blot analysis. Chromatin immunoprecipitation (ChIP) and luciferase assays were performed to observe the regulation of TRPM7 promoter regions by HIF-1α. A PCR array was utilized to screen glucose metabolism-related enzymes in HEK293 cells overexpressing TRPM7 induced by tetracycline, and then verified in TRPM7-overexpressed huh7 cells. Finally, CCK-8, transwell, scratch and tumor formation experiments in nude mice were conducted to examine the effect of TRPM7 on proliferation and metastasis in HCC. ResultsExposure to hypoxia led to increase the levels of TRPM7 and HIF-1α in HCC cells, which were inhibited by HIF-1α siRNA or enhanced by HIF-1α overexpression. HIF-1α directly bound to two hypoxia response elements (HREs) in the TRPM7 promoter. Several glycolytic metabolism-related enzymes, were simultaneously upregulated in HEK293 and huh7 cells overexpressing TRPM7 during hypoxia. In vitro and in vivo experiments demonstrated that TRPM7 promoted the proliferation and metastasis of HCC cells. ConclusionsTRPM7 was directly transcriptionally regulated by HIF-1α, leading to glycolytic metabolic reprogramming and the promotion of HCC proliferation and metastasis in vitro and in vivo. Our findings suggest that TRPM7 might be a potential diagnostic indicator and therapeutic target for HCC.

Wnt7a Decreases Brain Endothelial Barrier Function Via β-Catenin Activation

The blood-brain barrier consists of tightly connected endothelial cells protecting the brain’s microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/β-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated β-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/β-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of β-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by β-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by β-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of β-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease.

Effect of gold nanocomposite bearing HIF-1α siRNA on radiotherapy in nasopharyngeal carcinoma cell

Purpose: To investigate the effect of gold nanocomposite carrying hypoxia-inducible factor-1α (HIF-1α) siRNA on radiosensitivity of nasopharynx cancer (NPC) cells.
 Methods: Gold nanocomposite (AuNRs-(PEI-PEG)/RGD) or (AuPPR) was first synthesized and its cytotoxicity was evaluated. Then, the expression of HIF-1α in hypoxic NPC cells was assessed. In addition, the radiation sensitivity of AuPPR bearing HIF-1α siRNA on the cells was examined under xray exposure.
 Result: Gold nanocomposite (AuPPR) carrying HIF-1α siRNA (AuPPR-HIF-1α siRNA) significantly down-regulated HIF-1α expression. Under irradiation treatment, AuPPR-HIF-1α siRNA significantly enhanced apoptosis of NPC cells by 33.76 ± 3.65 %, when compared to the control and simple AuPPR group (22.5 ± 4.16 %, p < 0.01). Furthermore, cell cycle was significantly blocked in the sub-G1 phase in AuPPR-HIF-1α siRNA radiotherapy groups, indicating severe cell damage.
 Conclusion: This study has demonstrated that AuPPR-HIF-1α siRNA is effective in improving the radiosensitivity of nasopharyngeal carcinoma cells under hypoxia, and therefore can potentially be used to improve the radiotherapy of this carcinoma

Semisynthesis and anti-cancer properties of novel honokiol derivatives in human nasopharyngeal carcinoma CNE-2Z cells

In this study, 21 new honokiol derivatives were synthesised, and their anti-cancer properties were investigated. Among these, compound 1g exhibited the most potent cytotoxic activity against human nasopharyngeal carcinoma CNE-2Z cells, human gastric cancer SGC7901 cells, human breast cancer MCF-7 cells, and mouse leydig testicular cancer I-10 lines with IC50 values of 6.04, 7.17, 6.83, and 5.30 μM, respectively. Compared to the parental compound, 1g displayed up to 5.18-fold enhancement of the cytotoxic effect on CNE-2Z cells. We further demonstrated that 1g inhibited cell growth, suppressed migration and invasion, and induced apoptosis of CNE-2Z cells by down-regulating HIF-1α, MMP-2, MMP-9, Bcl-2, Akt and up-regulating Bax protein levels. Transfection of CNE-2Z cells with HIF-1α siRNA reduced cell migration and invasion. In addition, in vivo experiments confirmed that 1g inhibited tumour growth in CNE-2Z cell-xenografted nude mice with low toxicity. Thus, our data suggested that 1g was a potent and safe lead compound for nasopharyngeal carcinoma therapy.

Cav3.2 T-Type calcium channels downregulation attenuates bone cancer pain induced by inhibiting IGF-1/HIF-1α signaling pathway in the rat spinal cord

BackgroundBone cancer pain (BCP) is one of the most ubiquitous and refractory symptoms of cancer patients that needs to be urgently addressed. Substantial studies have revealed the pivotal role of Cav3.2 T-type calcium channels in chronic pain, however, its involvement in BCP and the specific molecular mechanism have not been fully elucidated. MethodsThe expression levels of Cav3.2, insulin-like growth factor 1(IGF-1), IGF-1 receptor (IGF-1R) and hypoxia-inducible factor-1α (HIF-1α) were detected by Western blot in tissues and cells. X-ray and Micro CT used to detect bone destruction in rats. Immunofluorescence was used to detect protein expression and spatial location in the spinal dorsal horn. Electrophoretic mobility shift assay used to verify the interaction between HIF-1α and Cav3.2. ResultsThe results showed that the expression of Cav3.2 channel was upregulated and blockade of this channel alleviated mechanical allodynia and thermal hyperalgesia in BCP rats. Additionally, inhibition of IGF-1/IGF-1R signaling not only reversed the BCP-induced upregulation of Cav3.2 and HIF-1α, but also decreased nociceptive hypersensitivity in BCP rats. Inhibition of IGF-1 increased Cav3.2 expression levels, which were abolished by pretreatment with HIF-1α siRNA in PC12 cells. Furthermore, nuclear HIF-1α bound to the promoter of Cav3.2 to regulate the Cav3.2 transcription level, and knockdown of HIF-1α suppresses the IGF-1-induced upregulation of Cav3.2 and pain behaviors in rats with BCP. ConclusionThese findings suggest that spinal Cav3.2 T-type calcium channels play a central role during the development of bone cancer pain in rats via regulation of the IGF-1/IGF-1R/HIF-1α pathway.

Macrophage-Cancer Hybrid Membrane-Camouflaged Nanoplatforms for HIF-1α Gene Silencing-Enhanced Sonodynamic Therapy of Glioblastoma.

Fabrication of ingenious nanomedicines to penetrate the blood-brain barrier (BBB) and blood-brain-tumor barrier (BBTB) for efficient glioblastoma (GBM) therapy remains a big challenge. In this work, macrophage-cancer hybrid membrane-camouflaged nanoplatforms were fabricated for target gene silencing-enhanced sonodynamic therapy (SDT) of GBM. The J774.A.1 macrophage cell membrane and the U87 glioblastoma cell membrane were fused to create a hybrid biomembrane (JUM) with good BBB penetration and glioblastoma targeting capability for camouflaging. The ZIF-8 nanoparticles were synthesized for indocyanine green (ICG) and HIF-1α siRNA encapsulation (ICG-siRNA@ZIF-8, ISZ) with a high loading efficiency. After accumulation in the tumor sites, the pH sensitivity of the nanoplatform enabled release of ICG and HIF-1α siRNA in the tumor cells. Then, the expression of HIF-1α could be efficiently inhibited by the released HIF-1α siRNA to increase the SDT efficiency under hypoxic conditions. In vitro and in vivo experiments revealed that ISZ@JUM showed good BBB penetration and brain tumor-targeting capability and could achieve effective gene silencing-enhanced SDT, demonstrating great promise for clinical applications.

#5453 LIPID NEPHROTOXICITY INDUCED BY HIF-1α ACTIVATION ACCELERATES INTERSTITIAL INJURY IN DIABETIC NEPHROPATHY

Abstract Background and Aims Our previous study demonstrated lipid accumulation contributes to tubulointerstitial injury in diabetic nephropathy. However, the exact mechanism of which accelerate lipid accumulation in tubulointerstitium has not been completely elucidated. Increasing studies suggest that chronic hypoxia plays an important pathophysiologic role in diabetic nephropathy. This study aimed to investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation induced by chronic hypoxia on lipid accumulation in diabetic kidney and to explore its potential mechanism. Method Human proximal tubular cell line HK-2 was incubated with or without cobalt chloride (CoCl2, 100 μM) which stimulates hypoxic condition in the presence or absence of glucose (30 mM) for 24 h. Specific siRNA was transfected in cells to knock down HIF-1α expression. Type 1 diabetic rats model were induced by STZ injection, and Lificiguat (YC-1) was used as HIF-1α inhibitor by intraperitoneal injection. Immunofluorescence staining and Western blot were used to determine the cellular localization and expression of HIF-1α. Periodic Acid-Schiff (PAS) staining was used to observe basic structure and pathological changes of kidney. The expression of carnitine palmitoyl transferase 1A (CPT1A), tissue growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) were measured by immunohistochemical staining and Western blot. Lipid accumulation was detected by oil red O staining and cellular triglyceride quantification assay. Results In in vitro study, CoCl2 effectively increased the expression of HIF-1α and its translocation from cytoplasm to nucleus. Meanwhile, HIF-1α activation induced by CoCl2 significantly increased lipid accumulation in HK-2 cells. Moreover, CoCl2 treatment increased TGF- β and CTGF expression in HK-2 cells. The presence of glucose (30mM) made no significant difference to HK-2 cells incubated with CoCl2. HIF-1α siRNA treatment significantly reduced lipid deposition in HK-2 cells incubated with CoCl2. In vivo study showed YC-1 treatment decreased the expression of HIF-1α in renal tubules of diabetic rats, which was accompanied by less lipid accumulation compared with the diabetes mellitus (DM) group. PAS staining showed the desquamation and necrosis of tubular epithelial cells in the YC-1 group were alleviated compared with the DM group. YC-1 treatment did not affect the ratio of kidney weight to body weight, whereas decreased the levels of blood glucose, urine albumin creatinine ratio and NAG creatinine ratio in diabetic rats. The expressions of fibrosis factor TGF- β and CTGF were decreased in the YC-1 group compared with the DM group. Furthermore, the protein expression of CPT1A, the key enzyme of fatty acid oxidation, which was decreased in the DM group compared with the controls, was regained by YC-1 treatment. Conclusion Our findings demonstrated that HIF-1α activation contributed to interstitial injury in diabetic nephropathy by inducing lipid accumulation. HIF-1α activation might contribute to lipid nephrotoxicity by downregulating fatty acid oxidation.

Puerarin-Tanshinone IIA Suppresses atherosclerosis inflammatory plaque via targeting succinate/HIF-1α/IL-1β axis

Ethnopharmacological relevanceInflammatory injury is an important pathological factor for the formation of atherosclerotic plaque. It is well known that Puerarin and Tanshinone IIA (Pue-Tan) can significantly reduce interleukin-1β (IL-1β) levels and delay the atherosclerosis (AS) process clinically in China. Previous evidence has shown that the Succinate/HIF-1α/IL-1β inflammatory signaling axis (Succinate axis) promotes the progression of atherosclerotic inflammatory plaques. It is not clear whether Pue-Tan inhibits inflammatory plaques by reducing the level of IL-1β through the succinate signaling axis. Aim of studyFind out the interaction between Pue-Tan targets and the succinate axis by means of network pharmacology and bioinformatics analysis and to further confirm whether Pue-Tan can inhibit vascular inflammation and delay the formation of atherosclerotic inflammatory plaques by targeting the succinate signaling axis. Materials and methodsFirstly, animal experiments were conducted to verify the changing relationship between Succinate and IL-1β under Pue-Tan intervention. Secondly, network pharmacology approach was employed to uncover the specific targets of Pue-Tan in the intervention of AS from multiple levels of components, proteins, and pathways, and at the same time, the target must be a key factor of the succinate signaling axis. Autodock vina1.5.6 was applied to molecular docking for Pue-Tan and target protein. Subsequently, cells experiment and animal experiment were performed to verify Pue-Tan inhibiting the inflammatory progression of atherosclerosis by targeting succinate signaling axis. ResultsFirstly, we first found that the reduction of IL-1β was positively correlated with succinate in the serum of Pue-Tan-treated mice. Secondly, network pharmacology compared with molecular docking showed that hypoxia-induced factor-1α (HIF-1α) was the key target of Pue-Tan and the key node of succinate singling axis. Finally, in vitro study, Pue-Tan significantly reduced the factors of succinate axis just as HIF-1α siRNA; in vivo study, we confirmed a decreased expression of succinate axis and ICAM-1 in the aorta of ApoE−/− mice under Pue-Tan intervention, which was consistent with the in vitro results. ConclusionThis study confirmed that Pue-Tan blocked the succinate axis by targeting HIF-1α to prevent the formation of atherosclerotic inflammatory plaques and delay the pathological process of AS. Network Pharmacology, Bioinformatics of Molecular Docking, and Molecular Biology Validation can be used as a effective way to discover and verify the pharmacological mechanism of TCM.

O093 Manipulating the hypoxia induced radioresistance in rectal cancer

Abstract Introduction Locally advanced rectal cancer is primarily treated with radiotherapy and chemotherapy. Tumour hypoxia induces strong radioresistance, although the reasons for this characteristic are still unclear. This research explores the mechanistic and biological factors underlying hypoxia-induced radioresistance, and development of novel therapeutic strategies to overcome this. Methods HCT116 and HT29 CRC cell lines were incubated in normoxia (21% O2) and hypoxic conditions (1% O2). Cells were irradiated at varying dosages of x-ray radiation before undergoing clonogenic survival assay, comet assay to assess DNA double strand break damage and repair, or immunobloting to investigate cell death-related factors. HIF1α and HIF1b siRNA knockdowns were used to assess radioresponse in hypoxia. Metformin was used to treat cells prior to radiation and assess cell survival. Results Hypoxia induces strong radioresistance in CRC cells compared to relative normoxia in clonogenic survival assays. Neutral comet assays showed no significant difference in double strand break (DSB) repair kinetics following irradiation in hypoxia compared to normoxia. Immunoblotting analysis demonstrates activation of hypoxia-inducible factor 1a (HIF1a) in hypoxia but not in normoxia. HIF1α and HIF1b siRNA knockdowns did not show any improvement in radioresistance. Metformin did not improve radiosensitivity of CRC cells under different oxygen conditions. Conclusion Hypoxia induces strong radioresistance in cultured CRC cells which is not explained by x-ray-induced DSB damage and the efficiency of its repair. HIF1a is activated in hypoxia alone and may contribute to this resistance. Metformin alone is unable to overcome this resistance and other therapeutic targets need to be explored.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Ginkgo biloba extract attenuates cisplatin-induced renal interstitial fibrosis by inhibiting the activation of renal fibroblasts through down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells

BackgroundActivation of renal fibroblasts into myofibroblasts plays an important role in promoting renal interstitial fibrosis (RIF). Ginkgo biloba extract (EGb) can alleviate RIF induced by cisplatin (CDDP). PurposeTo elucidate the effect of EGb treatment on cisplatin-induced RIF and reveal its potential mechanism. MethodsThe two main active components in EGb were determined by high-performance liquid chromatography (HPLC) analysis. Rats were induced by CDDP and then treated with EGb, 2ME2 (HIF-1α inhibitor) or amifostine. After HK-2 cells and HIF-1α siRNA HK-2 cells were treated with CDDP, EGb or amifostine, the conditioned medium from each group was cultured with NRK-49F cells. The renal function of rats was detected. The renal damage and fibrosis were evaluated by H&E and Masson trichrome staining. The IL-6 content in the cell medium was detected by ELISA. The expression levels of indicators related to renal fibrosis and signaling pathway were examined by western blotting and qRT-PCR. ResultsHPLC analysis showed that the contents of quercetin and kaempferol in EGb were 36.0 μg/ml and 45.7 μg/ml, respectively. In vivo, EGb and 2ME2 alleviated renal damage and fibrosis, as well as significantly decreased the levels of α-SMA, HIF-1α, STAT3 and IL-6 in rat tissues induced by CDDP. In vitro, the levels of HIF-1α, STAT3 and IL-6 were significantly increased in HK-2 cells and HIF-1α siRNA HK-2 cells induced by CDDP. Notably, HIF-1α siRNA significantly decreased the levels of HIF-1α, STAT3 and IL-6 in HK-2 cells, as well as the IL-6 level in medium from HK-2 cells. Additionally, the α-SMA level in NRK-49F cells was significantly increased after being cultured with conditioned medium from HK-2 cells or HIF-1α siRNA HK-2 cells exposed to CDDP. Furthermore, exogenous IL-6 increased the α-SMA level in NRK-49F cells. Importantly, the expression levels of the above-mentioned indicators were significantly decreased after the HK-2 cells and HIF-1α siRNA HK-2 cells were treated with EGb. ConclusionThis study revealed that EGb improves CDDP-induced RIF, and the mechanism may be related to its inhibition of the renal fibroblast activation by down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells.

Stellate ganglion block ameliorated central post-stroke pain with comorbid anxiety and depression through inhibiting HIF-1α/NLRP3 signaling following thalamic hemorrhagic stroke

BackgroundCentral post-stroke pain (CPSP) is an intractable and disabling central neuropathic pain that severely affects patients’ lives, well-being, and socialization abilities. However, CPSP has been poorly studied mechanistically and its treatment remains challenging. Here, we used a rat model of CPSP induced by thalamic hemorrhage to investigate its underlying mechanisms and the effect of stellate ganglion block (SGB) on CPSP and emotional comorbidities.MethodsThalamic hemorrhage was produced by injecting collagenase IV into the ventral-posterolateral nucleus (VPL) of the right thalamus. The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Behavioral tests were carried out to examine depressive and anxiety-like behaviors including the open field test (OFT), elevated plus maze test (EPMT), novelty-suppressed feeding test (NSFT), and forced swim test (FST). The peri-thalamic lesion tissues were collected for immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA). Genetic knockdown of thalamic hypoxia-inducible factor-1α (HIF-1α) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) with microinjection of HIF-1α siRNA and NLRP3 siRNA into the VPL of thalamus were performed 3 days before collagenase injection into the same regions. Microinjection of lificiguat (YC-1) and MCC950 into the VPL of thalamus were administrated 30 min before the collagenase injection in order to inhibited HIF-1α and NLRP3 pharmacologically. Repetitive right SGB was performed daily for 5 days and laser speckle contrast imaging (LSCI) was conducted to examine cerebral blood flow.ResultsThalamic hemorrhage caused persistent mechanical allodynia and anxiety- and depression-like behaviors. Accompanying the persistent mechanical allodynia, the expression of HIF-1α and NLRP3, as well as the activities of microglia and astrocytes in the peri-thalamic lesion sites, were significantly increased. Genetic knockdown of thalamic HIF-1α and NLRP3 significantly attenuated mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. Further studies revealed that intra-thalamic injection of YC-1, or MCC950 significantly suppressed the activation of microglia and astrocytes, the release of pro-inflammatory cytokines, the upregulation of malondialdehyde (MDA), and the downregulation of superoxide dismutase (SOD), as well as mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage. In addition, repetitive ipsilateral SGB significantly restored the upregulated HIF-1α/NLRP3 signaling and the hyperactivated microglia and astrocytes following thalamic hemorrhage. The enhanced expression of pro-inflammatory cytokines and the oxidative stress in the peri-thalamic lesion sites were also reversed by SGB. Moreover, LSCI showed that repetitive SGB significantly increased cerebral blood flow following thalamic hemorrhage. Most strikingly, SGB not only prevented, but also reversed the development of mechanical allodynia and anxiety- and depression-like behaviors induced by thalamic hemorrhage. However, pharmacological activation of thalamic HIF-1α and NLRP3 with specific agonists significantly eliminated the therapeutic effects of SGB on mechanical allodynia and anxiety- and depression-like behaviors following thalamic hemorrhage.ConclusionThis study demonstrated for the first time that SGB could improve CPSP with comorbid anxiety and depression by increasing cerebral blood flow and inhibiting HIF-1α/NLRP3 inflammatory signaling.

Hypoxia-inducible factor 1α inhibitor induces cell death via suppression of BCR-ABL1 and Met expression in BCR-ABL1 tyrosine kinase inhibitor sensitive and resistant chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) has a markedly improved prognosis with the use of breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitors (BCR-ABL1 TKIs). However, approximately 40% of patients are resistant or intolerant to BCR-ABL1 TKIs. Hypoxia-inducible factor 1α (HIF-1α) is a hypoxia response factor that has been reported to be highly expressed in CML patients, making it a therapeutic target for BCR-ABL1 TKI-sensitive CML and BCR-ABL1 TKI-resistant CML. In this study, we examined whether HIF-1α inhibitors induce cell death in CML cells and BCR-ABL1 TKI-resistant CML cells. We found that echinomycin and PX-478 induced cell death in BCR-ABL1 TKIs sensitive and resistant CML cells at similar concentrations while the cell sensitivity was not affected with imatinib or dasatinib in BCR-ABL1 TKIs resistant CML cells. In addition, echinomycin and PX-478 inhibited the c-Jun N-terminal kinase (JNK), Akt, and extracellular-regulated protein kinase 1/2 (ERK1/2) activation via suppression of BCR-ABL1 and Met expression in BCR-ABL1 sensitive and resistant CML cells. Moreover, treatment with HIF-1α siRNA induced cell death by inhibiting BCR-ABL1 and Met expression and activation of JNK, Akt, and ERK1/2 in BCR-ABL1 TKIs sensitive and resistant CML cells. These results indicated that HIF-1α regulates BCR-ABL and Met expression and is involved in cell survival in CML cells, suggesting that HIF-1α inhibitors induce cell death in BCR-ABL1 TKIs sensitive and resistant CML cells and therefore HIF-1α inhibitors are potential candidates for CML treatment. [BMB Reports 2023; 56(2): 78-83].

A pH-responsive metal-organic framework for the co-delivery of HIF-2α siRNA and curcumin for enhanced therapy of osteoarthritis

The occurrence of osteoarthritis (OA) is highly correlated with the reduction of joint lubrication performance, in which persistent excessive inflammation and irreversible destruction of cartilage dominate the mechanism. The inadequate response to monotherapy methods, suboptimal efficacy caused by undesirable bioavailability, short retention, and lack of stimulus-responsiveness, are few unresolved issues. Herein, we report a pH-responsive metal-organic framework (MOF), namely, MIL-101-NH2, for the co-delivery of anti-inflammatory drug curcumin (CCM) and small interfering RNA (siRNA) for hypoxia inducible factor (HIF-2α). CCM and siRNA were loaded via encapsulation and surface coordination ability of MIL-101-NH2. Our vitro tests showed that MIL-101-NH2 protected siRNA from nuclease degradation by lysosomal escape. The pH-responsive MIL-101-NH2 gradually collapsed in an acidic OA microenvironment to release the CCM payloads to down-regulate the level of pro-inflammatory cytokines, and to release the siRNA payloads to cleave the target HIF-2α mRNA for gene-silencing therapy, ultimately exhibiting the synergetic therapeutic efficacy by silencing HIF-2α genes accompanied by inhibiting the inflammation response and cartilage degeneration of OA. The hybrid material reported herein exhibited promising potential performance for OA therapy as supported by both in vitro and in vivo studies and may offer an efficacious therapeutic strategy for OA utilizing MOFs as host materials.