BM-MSC-Loaded Graphene-Collagen Cryogels Ameliorate Neuroinflammation in a Rat Spinal Cord Injury Model.

A major obstacle to axonal regeneration following spinal cord injury (SCI) is neuroinflammation mediated by astrocytes and microglial cells. We previously demonstrated that graphene-based collagen hydrogels alone can decrease neuroinflammation in SCI. Their regenerative potential, however, is poorly understood and incomplete. Furthermore, stem cells have demonstrated both neuroprotective and regenerative properties in spinal cord regeneration, although there are constraints connected with the application of stem cell-based therapy. In this study, we have analyzed the regeneration capability of human bone marrow mesenchymal stem cell (BM-MSC)-loaded graphene-cross-linked collagen cryogels (Gr-Col) in a thoracic (T10-T11) hemisection model of SCI. Our study found that BM-MSC-loaded Gr-Col improves axonal regeneration, reduces neuroinflammation by decreasing astrocyte reactivity, and promotes M2 macrophage polarization. BM-MSC-loaded-Gr-Col demonstrated enhanced regenerative potential compared to Gr-Col and the injury group control. Next-generation sequencing (NGS) analysis revealed that BM-MSC-loaded-Gr-Col modulates the JAK2-STAT3 pathway, thus decreasing the reactive and scar-forming astrocyte phenotype. The decrease in neuroinflammation in the BM-MSC-loaded-Gr-Col group is attributed to the modulation of Notch/Rock and STAT5a/b and STAT6 signaling. Overall, Gene Set Enrichment Analysis suggests the promising role of BM-MSC-loaded-Gr-Col in promoting axonal regeneration after SCI by modulating molecular pathways such as the PI3/Akt pathway, focal adhesion kinase, and various inflammatory pathways.

Mechanistic Investigation of Thiazole-Based Pyruvate Kinase M2 Inhibitor Causing Tumor Regression in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a deadly breast cancer with a poor prognosis. Pyruvate kinase M2 (PKM2), a key rate-limiting enzyme in glycolysis, is abnormally highly expressed in TNBC. Overexpressed PKM2 amplifies glucose uptake, enhances lactate production, and suppresses autophagy, thereby expediting the progression of oncogenic processes. A high mortality rate demands novel chemotherapeutic regimens at once. Herein, we report the rational development of an imidazopyridine-based thiazole derivative 7d as an anticancer agent inhibiting PKM2. Nanomolar range PKM2 inhibitors with favorable drug-like properties emerged through enzyme assays. Experiments on two-dimensional (2D)/three-dimensional (3D) cell cultures, lactate release assay, surface plasmon resonance (SPR), and quantitative real-time polymerase chain reaction (qRT-PCR) validated 7d preclinically. In vivo, 7d outperformed lapatinib in tumor regression. This investigation introduces a lead-based approach characterized by its clear-cut chemistry and robust efficacy in designing an exceptionally potent inhibitor targeting PKM2, with a focus on combating TNBC.

MiR-29b modulates DNA methylation in promoter region of miR-130b in mouse model of Diabetic nephropathy.

Epigenetic modifications play a role in Diabetic Nephropathy (DN). Downregulation of miR-29b leads to modulation of DNA methylation via DNA methyl transferases (DNMTs) and hence exaggerated renal fibrosis in DN. Therefore, the main aim of the study was to evaluate effect of miR-29b expression in vivo on DNMTs, renal fibrosis, glomerular and tubular damage as well as renal morphology in DN. In order to explore the role of miR-29b in DNA methylation of other miRNAs, methylation profiling study was performed. It revealed that miR-29b was involved in methylation on of miR-130b on the cytosine guanine dinucleotides rich DNA (CpG) island 1 located on promoter region. In conclusion, miR-29b expression was found to modulate DNA methylation via DNMTs and regulate methylation of miR-130b. The result of this study provides a future direction to unveil role of miRNA expression in DNA methylation and its consequent effect on other miRNAs in DN. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01208-2.

Hyaluronic acid: More than a carrier, having an overpowering extracellular and intracellular impact on cancer.

Hyaluronic acid (HA), also named hyaluronan, is an omnipresent component of the tissue microenvironment. It is extensively used to formulate targeted drug delivery systems for cancer. Although HA itself has pivotal influences in various cancers, its calibers are somewhat neglected when using it as delivering platform to treat cancer. In the last decade, multiple studies revealed roles of HA in cancer cell proliferation, invasion, apoptosis, and dormancy through pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK), P38, and nuclear factor kappa-light chain-enhancer of activated B cells (NFκB). A more fascinating fact is that the distinct molecular weight (MW) of HA exerts disparate effects on the same type of cancer. Its overwhelming use in cancer therapy and other therapeutic products make collective research on the sundry impact of it on various types of cancer, an essential aspect to be considered in all of these domains. Even the development of new therapies against cancer needed meticulous studies on HA because of its divergence of activity based on MW. This review will provide painstaking insight into the extracellular and intracellular bioactivity of HA, its modified forms, and its MW in cancers, which may improve the management of cancer.

Polystyrene-Based Slippery Surfaces Enable the Generation and Easy Retrieval of Tumor Spheroids.

Multicellular tumor spheroids are the most well-characterized organotypic models for cancer research. Generally, scaffold-based and scaffold-free techniques are widely used for culturing spheroids. In scaffold-free techniques, the hanging drop (HD) method is a more versatile technique, but the retrieval of three-dimensional (3D) cell spheroids in the hanging drop method is usually labor-intensive. We developed oil-coated polystyrene nanofiber-based reusable slippery surfaces for the generation and easy retrieval of 3D spheroids. The developed slippery surfaces facilitated the rolling and gliding of the cell medium drops as well as holding the hydrophilic drops for more than 72 h by the virtue of surface tension as in the hanging drop method. In this study, polystyrene nanofibers were developed by the facile technique of electrospinning and the morphological evaluation was performed by scanning electron microscopy (SEM) and cryo-FESEM. We modeled the retrieval process of 3D spheroids with the ingredients of 3D spheroid generation, such as water, cell culture media, collagen, and hyaluronic acid solution, demonstrating the faster and easy retrieval of 3D spheroids within a few seconds. We created MCF-7 spheroids as a proof of concept with a developed slippery surface. 3D spheroids were characterized for their size, homogeneity, reactive oxygen species, proliferative marker (Ki-67), and hypoxic inducing factor 1ά (HIF-1ά). These 3D tumor spheroids were further tested for evaluating the cellular toxicity of the doxorubicin drug. Hence, the proposed slippery surfaces demonstrated the potential alternative of culturing 3D tumor spheroids with an easy retrieval process with intact 3D spheroids.

Extrinsic hyaluronic acid induction differentially modulates intracellular glutamine metabolism in breast cancer stem cells.

The effect of low and high molecular weight hyaluronic acid on glutamine metabolism in luminal and basal breast cancer and cancer stem cells is being investigated. In luminal cell lines (MCF-7), HA enhances the intracellular utilization of gln in redox metabolism and decreases its use in TCA. On the contrary, in MDAMB-231 cells, HA induces the uptake of gln to be utilized in anaplerosis rather than ROS maintenance. However, in MCF-7 CSCs, HA induces up-regulation of xCT, further, it uses gln-derived glutamate for the exchange of cystine, thus maintaining ROS levels through xCT. MDA-MB-231 CSCs reduce the secretion of glutamate in response to HA and decrease the gln flux towards reductive carboxylation. Conclusively, our study demonstrated that although the uptake of gln is enhanced by HA, it is differentially utilized intracellularly in breast cancer cells. This study could significantly influence the therapeutics involving HA and Gln in breast cancer.

Graphene-collagen cryogel controls neuroinflammation and fosters accelerated axonal regeneration in spinal cord injury.

Spinal cord injury (SCI) is a devastating condition resulting in loss of motor function. The pathology of SCI is multifaceted and involves a cascade of events, including neuroinflammation and neuronal degeneration at the epicenter, limiting repair process. We developed a supermacroporous, mechanically elastic, electro-conductive, graphene crosslinked collagen (Gr-Col) cryogels for the regeneration of the spinal cord post-injury. The effects of graphene in controlling astrocytes reactivity and microglia polarization are evaluated in spinal cord slice organotypic culture and rat spinal cord lateral hemisection model of SCI. In our work, the application of external electric stimulus results in the enhanced expression of neuronal markers in an organotypic culture. The implantation of Gr-Col cryogels in rat thoracic T9-T11 hemisection model demonstrates an improved functional recovery within 14 days post-injury (DPI), promoted myelination, and decreases the lesion volume at the injury site. Decrease in the expression of STAT3 in the implanted Gr-Col cryogels may be responsible for the decrease in astrocytes reactivity. Microglia cells within the implanted cryogels shows higher anti-inflammatory phenotype (M2) than inflammatory (M1) phenotype. The higher expression of mature axonal markers like ß-tubulin III, GAP43, doublecortin, and neurofilament 200 in the implanted Gr-Col cryogel confirms the axonal regeneration after 28 DPI. Gr-Col cryogels also modulate the production of ECM matrix, favouring the axonal regeneration. This study shows that Gr-Col cryogels decreases neuroinflammation and accelerate axonal regeneration.

X-ray tomography analysis reveals the influence of graphene on porous morphology of collagen cryogels.

X-ray micro-tomography based analysis of porous hydrogel has gained a wide attention recently. It provides an advantage in three-dimensional analysis of pore morphometric and interconnection within the hydrogel network. We have fabricated highly elastic graphene crosslinked collagen hydrogel using cryogelation technique. The influence of graphene as a nano-crosslinker on the overall porosity and inter-connections between the pores in collagen cryogels was determined using X-ray micro-tomography. We have evaluated the effect of different concentration of amino-functionalized graphene nano-crosslinker on collagen cryogels porosity, pore volume, interconnectivity density, fractal dimensions and pore wall thickness. This study, reveals that the use of graphene as a nano-crosslinker have improved micro-architecture as compare to collagen cryogels in the absence of graphene for tissue engineering applications.

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein-protein interactions.

Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer's disease, allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, sepsis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejections. Despite several expansions in our understanding of inflammatory disorders and their mediators, it seems clear that numerous proteins participate in the onset of CI. One crucial protein pyruvate kinase M2 (PKM2) much studied in cancer is also found to be inextricably woven in the onset of several CI's. It has been found that PKM2 plays a significant role in several disorders using a network of proteins that interact in multiple ways. For instance, PKM2 forms a close association with epidermal growth factor receptors (EGFRs) for uncontrolled growth and proliferation of tumor cells. In neurodegeneration, PKM2 interacts with apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) to onset Alzheimer's disease pathogenesis. The cross-talk of protein tyrosine phosphatase 1B (PTP1B) and PKM2 acts as stepping stones for the commencement of diabetes. Perhaps PKM2 stores the potential to unlock the pathophysiology of several diseases. Here we provide an overview of the notoriously convoluted biology of CI's and PKM2. The cross-talk of PKM2 with several proteins involved in stroke, Alzheimer's, cancer, and other diseases has also been discussed. We believe that considering the importance of PKM2 in inflammation-related diseases, new options for treating various disorders with the development of more selective agents targeting PKM2 may appear.

Discovery of boronic acid-based potent activators of tumor pyruvate kinase M2 and development of gastroretentive nanoformulation for oral dosing.

Several studies have established that cancer cells explicitly over-express the less active isoform of pyruvate kinase M2 (PKM2) is critical for tumorigenesis. The activation of PKM2 towards tetramer formation may increase affinity towards phosphoenolpyruvate (PEP) and avoidance of the Warburg effect. Herein, we describe the design, synthesis, and development of boronic acid-based molecules as activators of PKM2. The designed molecules were inspired by existing anticancer scaffolds and several fragments were assembled in the derivatives. 6a-6d were synthesized using a multi-step synthetic strategy in 55-70% yields, starting from cheap and readily available materials. The compounds were selectively cytotoxic to kill the cancerous cells at 80 nM, while they were non-toxic to the normal cells. The kinetic studies established the compounds as novel activators of PKM2 and (E/Z)-(4-(3-(2-((4-chlorophenyl)amino)-4-(dimethylamino)thiazol-5-yl)-2-(ethoxycarbonyl)-3-oxoprop-1-en-1-yl) phenyl)boronic acid (6c) emerged as the most potent derivative. 6c was further evaluated using various in silico tools to understand the molecular mechanism of tetramer formation. Docking studies revealed that 6c binds to the PKM2 dimer at the dimeric interface. Further to ascertain the binding site and mechanism of action, rigorous MD (molecular dynamics) simulations were undertaken, which led to the conclusion that 6c stabilizes the center of the dimeric interface that possibly promotes tetramer formation. We further planned to make a tablet of the developed molecule for oral delivery, but it was seriously impeded owing to poor aqueous solubility of 6c. To improve aqueous solubility and retain 6c at the lower gastrointestinal tract, thiolated chitosan-based nanoparticles (TCNPs) were prepared and further developed as tablet dosage form to retain anticancer potency in the excised goat colon. Our findings may provide a valuable pharmacological mechanism for understanding metabolic underpinnings that may aid in the clinical development of new anticancer agents targeting PKM2.

Design, synthesis and in-vitro evaluation of fluorinated triazoles as multi-target directed ligands for Alzheimer disease.

Alzheimer disease is multi-factorial and inflammation plays a major role in the disease progression and severity. Metals and reactive oxygen species (ROS) are the key mediators for inflammatory conditions associated with Alzheimer's. Along multi-factorial nature, major challenge for developing new drug is the ability of the molecule to cross blood brain barrier (BBB). We have designed and synthesized multi-target directed hexafluorocarbinol containing triazoles to inhibit Amyloid ß aggregation and simultaneously chelate the excess metals present in the extracellular space and scavenge the ROS thus reduce the inflammatory condition. From the screened compound library, compound 1c found to be potent and safe. It has demonstrated inhibition of Amyloid ß aggregation (IC50 of 4.6 µM) through selective binding with Amyloid ß at the nucleation site (evidenced from the molecular docking). It also chelate metals (Cu+2, Zn+2 and Fe+3) and scavenges ROS significantly. Due to the presence of hexafluorocarbinol moiety in the molecule it may assist to permeate BBB and improve the pharmacokinetic properties. The in-vitro results of compound 1c indicate the promiscuity for the development of hexafluorocarbinol containing triazoles amide scaffold as multi-target directed therapy against Alzheimer disease.

Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction.

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA-PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in 50 mM glucose compared to that of 10 mM glucose.

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine-deprivation.

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24+ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAMhigh population whereas MDAMB-231-bCSCs increased CD44high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of ß-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of ß-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.

Evaluation of Tissue Engineering Approaches for Intervertebral Disc Regeneration in Relevant Animal Models.

Translation of tissue engineering strategies for the regeneration of intervertebral disc (IVD) requires a strong understanding of pathophysiology through the relevant animal model. There is no relevant animal model due to differences in disc anatomy, cellular composition, extracellular matrix components, disc physiology, and mechanical strength from humans. However, available animal models if used correctly could provide clinically relevant information for the translation into humans. In this review, we have investigated different types of strategies for the development of clinically relevant animal models to study biomaterials, cells, biomolecular or their combination in developing tissue engineering-based treatment strategies. Tissue engineering strategies that utilize various animal models for IVD regeneration are summarized and outcomes have been discussed. The understanding of animal models for the validation of regenerative approaches is employed to understand and treat the pathophysiology of degenerative disc disease (DDD) before proceeding for human trials. These animal models play an important role in building a therapeutic regime for IVD tissue regeneration, which can serve as a platform for clinical applications.

A Polycaprolactone (PCL)-Supported Electrocompacted Aligned Collagen Type-I Patch for Annulus Fibrosus Repair and Regeneration.

Deformity or fissure within the annulus fibrosus (AF) lamellar structure often results in disc herniation leading to the extrusion of nucleus pulposus (NP), which pushes the adjacent nerve, causing low back pain. Low back pain, frequently associated with the degeneration of the intervertebral disc (IVD), affects around 80% of the population worldwide. The difficulty in mimicking the unique structural characteristics of the native AF tissue presents several challenges to the tissue engineering field for the development of the long-term effective therapeutic strategy for AF tissue regeneration. The AF cell niche possesses less reparative capacity for regeneration and thus compels to develop a strategy to recapitulate damaged AF tissues. We have fabricated a polycaprolactone-supported electrocompacted type-I collagen patch (A-PCL-NH2+Col-I) using surface-modified electrospun-aligned polycaprolactone (A-PCL) nanofibers cross-linked with an electro-compacted type-I collagen patch (Col-I) using EDAC-NHS (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride and N-hydroxy succinimide). This subtle approach offered a 3D biodegradable scaffold with dense aggregates of anisotropic collagen-I nanofibrils coupled with electrospun-aligned PCL nanofibers, which provide high tensile strength (4.21 ± 1.07 MPa), moduli (24.496 ± 4.85 MPa), low subsidence to failure, and high-water absorption ability. The systemic organization of both the polymers within the scaffold, evident from attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, revealed a uniform degree of fiber alignment assessed by differential interference contrast (DIC) microscopy, field-emission scanning electron microscopy (FE-SEM), and cryo-SEM. The aminolysis of A-PCL nanofibers was established by energy-dispersive X-ray analysis (EDX), while circular dichroic spectra showed that the electro-compacted Col-I patch displayed a triple helical structure, characteristic of collagens. Moreover, the scaffold revealed more hydrophilic, rough nano-features, which provided ample ligands for cell attachment supporting adequate proliferation of primary goat annulus fibrosus (AF) cells, oriented along the fiber direction, and also favored sufficient production of collagen type-I (+32-fold change) and a glycosaminoglycan extracellular matrix (+2.3-fold change) as compared to cell control, respectively. This study thus demonstrates for the first time the practicability of creating an aligned polycaprolactone-supported electrocompacted type-I collagen hydrogel (A-PCL-NH2+Col-I) with significant biomechanical properties, which can be used as an alternative to repair and regenerate AF fissures in degenerated IVD.

Highly elastic, electroconductive, immunomodulatory graphene crosslinked collagen cryogel for spinal cord regeneration.

Novel amino-functionalized graphene crosslinked collagen based nerve conduit having appropriate electric (3.8 ± 0.2 mSiemens/cm) and mechanical cues (having young modulus value of 100-347 kPa) for stem cell transplantation and neural tissue regeneration was fabricated using cryogelation. The developed conduit has shown sufficiently high porosity with interconnectivity between the pores. Raman spectroscopy analysis revealed the increase in orderliness and crosslinking of collagen molecules in the developed cryogel due to the incorporation of amino-functionalized graphene. BM-MSCs grown on graphene collagen cryogels have shown enhanced expression of CD90 and CD73 gene upon electric stimulation (100 mV/mm) contributing towards maintaining their stemness. Furthermore, an increased secretion of ATP from BM-MSCs grown on graphene collagen cryogel was also observed upon electric stimulation that may help in regeneration of neurons and immuno-modulation. Neuronal differentiation of BM-MSCs on graphene collagen cryogel in the presence of electric stimulus showed an enhanced expression of MAP-2 kinase and ß-tubulin III. Immunohistochemistry studies have also demonstrated the improved neuronal differentiation of BM-MSCs. BM-MSCs grown on electro-conductive collagen cryogels under inflammatory microenvironment in vitro showed high indoleamine 2,3 dioxygenase activity. Moreover, macrophages cells grown on graphene collagen cryogels have shown high CD206 (M2 polarization marker) and CD163 (M2 polarization marker) and low CD86 (M1 polarization marker) gene expression demonstrating M2 polarization of macrophages, which may aid in tissue repair. In an organotypic culture, the developed cryogel conduit has supported cellular growth and migration from adult rat spinal cord. Thus, this novel electro-conductive graphene collagen cryogels have potential for suppressing the neuro-inflammation and promoting the neuronal cellular migration and proliferation, which is a major barrier during the spinal cord regeneration.

Hyaluronic acid containing scaffolds ameliorate stem cell function for tissue repair and regeneration.

Recent evidence based studies have proposed hyaluronic acid (HA) as an emerging biopolymer for various tissue engineering application. Meanwhile, stem cells (SCs) have also gained immense popularity for their tissue regenerative capacity. Thus, combining HA and stem cells for tissue engineering application have shown to foster tissue repair and regeneration process. HA possesses the ability to interact with SCs via cellular surface receptors along with the capacity to elicit the process of differentiation. The influence of HA on stem cells has been widely investigated in cartilage and bone repair but their properties of reducing inflammation has also been explored in various other tissue repair processes. In this review, we have provided an insight to the effect of crosslinked and non-crosslinked HA on various stem cells. Further, HA based scaffolds combined with stem cells have shown to have a synergistic effect in the regeneration capacity. Also, various chemically modified HA and biomolecules conjugated HA as a suitable carrier or matrix for stem cells delivery and the effect of HA in fine tuning the stem cells function is discussed.

Target specific intracellular quantification of etoposide by quadrupole-time of flight based mass spectrometric method.

Etoposide (ETP), a widely used chemotherapeutic agent has an intracellular target site of action. Unfortunately, the concentration of ETP in plasma does not properly reflect the concentration in its intracellular site of action. As per our knowledge, no reported bioanalytical method is available for intracellular quantification of ETP. In this research, we developed an LC-MS/MS method to quantitate ETP in intracellular compartments of MCF-7 cells. The Abcam nuclear extraction kit was used for extracting the nuclear and cytosolic protein from MCF-7 cells. The method showed excellent linearity in the 20-1000 ng/mL range. The intra and inter-day precision (%CV) including LLOQ were found to be in the range of 2.19-16.96% and 6.71-11.21%, respectively, with an accuracy of 86.87 to 110.37% and 93.03 to 100.50%, respectively. The concentration of ETP in nuclear and cytosolic fraction was successfully quantitated using the developed method. The developed method can be applied to understand the efficacy of different formulations based on the intracellular ETP concentration in vitro. It can be considered as a model method for quantification of other similar categories of drugs in their actual intracellular site of action after required optimization in the methodology.

miR-29b attenuates histone deacetylase-4 mediated podocyte dysfunction and renal fibrosis in diabetic nephropathy.

PURPOSE: As epigenetic modifications like chromatin histone modifications have been suggested to play a role in the pathophysiology of Diabetic Nephropathy (DN) and are also found to be regulated by microRNAs. Our main purpose was to explore the role of microRNA in histone modulations associated with DN. There is downregulation of miR-29b due to advanced glycation end products in diabetes. Histone Deacetylase-4 (HDAC4) is amongst the histone modulators which promotes podocytes' impairment and upregulates transforming growth factor-1 (TGF-ß1) leading to renal fibrosis. Moreover, macrophage infiltration causes podocytes' apoptosis and IL-6 mediated inflammation. As miR-29b is downregulated in diabetes and HDAC4, TGF-ß1 and IL-6 could be the possible therapeutic targets in DN, our study was focussed on unveiling the role of miR-29b in modulation of HDAC4 and hence, in podocyte dysfunction and renal fibrosis in DN. METHODS: In silico analysis and luciferase assay were done to study the interaction between miR-29b and HDAC4. In-vitro DN model was developed in podocytes and miR-29b mimics were transfected. Also, podocytes were co-cultured with macrophage and miR-29b mimics were transfected. At the end, in-vivo DN model was generated in C57BL/6 J male mice and the effect of miR-29b mimics was reconfirmed. RESULTS: It was found that miR-29b targets the 3' untranslated region of HDAC4. In both in-vitro and in-vivo DN model, downregulation of miR-29b and subsequent increase in HDAC4 expression was observed. The miR-29b mimics suppressed podocytes' inflammation mediated through macrophages and attenuated HDAC4 expression, glomerular damage and renal fibrosis. CONCLUSION: This study concludes that miR-29b regulates the expression of HDAC4 which plays a role in controlling renal fibrosis and podocytes' impairment in DN.