Researchers first examined NGR1 in differentiated PC12 cells subjected to six hours of oxygen-glucose deprivation (OGD), a laboratory model of ischemic conditions. OGD reduced intracellular ATP and mitochondrial oxygen consumption while increasing extracellular acidification, lactate and lactate dehydrogenase release, indicating impaired mitochondrial function and increased glycolytic activity. NGR1 treatment, particularly at 10 μM, reduced cell death and apoptosis, restored ATP levels and mitochondrial membrane potential, and brought oxygen consumption and extracellular acidification close to baseline values.

The cellular analyses also identified changes in the CKLF1/CCR5 pathway. OGD increased CKLF1, CCR5, IL-1β, phosphorylated mTOR, HIF-1α, PFKFB3 and GLUT3, while AMPK phosphorylation decreased. NGR1 attenuated these changes, and immunofluorescence showed less CKLF1/CCR5/HIF-1α colocalization after treatment. The authors therefore associated NGR1 activity with suppression of the AMPK/mTOR/HIF-1α-linked metabolic shift toward glycolysis.

Computational and biochemical experiments were used to investigate how NGR1 might affect CKLF1. Molecular docking predicted a binding energy of −7.4 kcal/mol, and a 100-nanosecond molecular-dynamics simulation indicated a stable complex. Cellular thermal shift testing showed that 10 μM NGR1 increased CKLF1’s thermal denaturation temperature by 7.4 °C, while DARTS experiments showed reduced proteolysis of CKLF1 in treated cell lysates. These results supported a direct physical interaction, although binding-affinity constants were not measured.

In male Sprague-Dawley rats subjected to pMCAO, intraperitoneal NGR1 at 10, 20 or 40 mg/kg, administered six hours after ischemia, reduced infarct volume and cerebral edema, improved scores in several neurobehavioral tests and preserved neuronal morphology in the cortex and hippocampal CA1 region. NGR1 also reduced serum IL-1β and TNF-α and increased IL-4 and IL-10. Edaravone was used as a positive control, and NGR1 produced stronger suppression of CKLF1 immunoreactivity at equal doses in the reported comparisons.

Brain-tissue analyses showed that ischemia increased CKLF1 expression and the formation of CKLF1 complexes with CCR5 and HIF-1α in the hippocampus and cortex. NGR1 reduced these protein interactions and lowered the expression of glycolytic mediators, including PFKFB3 and GLUT3. These measurements were made in rat tissue, and the study used whole-brain homogenates that could not distinguish metabolic changes by cell type.

The contribution of CKLF1 was tested in additional male rats lacking the CKLF1 gene. Knockout rats had smaller infarcts, less edema and better neurological scores than wild-type ischemic rats. However, NGR1 did not provide additional improvement in infarct volume, edema, neurological function, neuronal morphology, inflammatory cytokines or the measured AMPK/mTOR/HIF-1α and glycolytic protein changes in knockout animals. Loss of CKLF1 therefore removed the measured protective effects of NGR1 in this model.

The authors noted an important limitation in the knockout results: ischemic metabolic changes in the AMPK/mTOR/HIF-1α pathway and glycolytic proteins remained similar in knockout and wild-type rats, even though knockout reduced injury. They consequently characterized CKLF1 as required for NGR1-mediated metabolic correction rather than the only pathway driving ischemic metabolic reprogramming.

Other limitations include the use of a neuron-like cell line that may not reproduce the metabolism of primary mature neurons, incomplete characterization of NGR1’s pharmacokinetic profile, and the short-term, preclinical nature of the rat experiments. The study reports a mechanistic relationship between NGR1, CKLF1 and neuronal injury in experimental models; it does not show that NGR1 treats ischemic stroke in patients.

Subject of Research: Preclinical study of NGR1 and CKLF1-dependent neuronal metabolic reprogramming after ischemic injury

Article Title: Notoginsenoside R1 attenuates ischemic stroke via CKLF1-dependent suppression of neuronal metabolic reprogramming

Article References: Wang, H., Liu, S., Chen, C., Long, J., Yan, Q., Lin, Y., Li, S., Huang, Y., Yang, S., Lin, M., Liu, X., Yi, F., Tan, Y., Chen, N., Ai, Q., & Yang, Y. (2026). Notoginsenoside R1 attenuates ischemic stroke via CKLF1-dependent suppression of neuronal metabolic reprogramming. Frontiers in Pharmacology, 17, Article 1964218. https://doi.org/10.3389/fphar.2026.1964218

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1964218

Keywords: notoginsenoside R1, ischemic stroke, CKLF1, CCR5, metabolic reprogramming, neuroprotection, PC12 cells, pMCAO rats, Notoginsenoside, attenuates, ischemic, stroke

The central obstacle to curing HIV is the latent reservoir: a population of CD4-expressing cells carrying transcriptionally silent but intact proviruses. Antiretroviral therapy (ART) suppresses plasma viraemia indefinitely but does not touch these cells. Cells with actively expressed provirus can be attacked by broadly neutralizing antibodies, but latently infected cells expose no viral proteins and therefore cannot be selectively targeted without first forcing the virus out of hiding. To date, the only interventions that have convincingly eliminated the reservoir are cytotoxic conditioning regimens and allogeneic stem-cell transplantation from donors carrying the CCR5-Δ32 mutation, which renders the receptor non-functional and blocks entry of most HIV strains.

Previous early-treatment experiments in macaques have been uniformly disappointing. When ART was initiated three or seven days after SIV or SHIV infection, no animals achieved remission. In one large study using a low-dose intravenous inoculum designed to model slow human transmission, only one of thirteen animals treated six days after infection failed to rebound, and none treated at seven days did. Even allogeneic stem-cell transplantation in Mauritian cynomolgus macaques, which can produce cure, is sometimes defeated by viral spread to engrafting CCR5-positive donor cells. The new study therefore asked a different question: rather than relying on ART alone, could the cells that harbour and sustain the early reservoir be physically removed?

The experimental design was deliberately aggressive. Twenty-five newborn rhesus macaques were randomized at birth, and at roughly two to three weeks of age they received a high oral dose of SIVmac251, a highly pathogenic and genetically heterogeneous swarm virus. The animals were left untreated for seven days to allow a permanent, systemic infection to establish, then placed on a triple antiretroviral regimen of tenofovir, emtricitabine and dolutegravir. Two days after ART initiation, one group continued on ART alone, a second received a CD4-depleting antibody called CD4R1, and a third received a single intravenous dose of an anti-CCR5/CD3 bispecific antibody, a molecule engineered to bind CCR5 on target cells and CD3 on T cells, thereby recruiting T cells to destroy any cell expressing CCR5. ART was withdrawn after eighteen weeks and viral rebound was monitored.

Twenty of the twenty-five infants were successfully infected, and all infected animals showed unambiguous systemic SIV infection with repeated plasma viral-load detections and cell-associated viral RNA. Both depleting antibodies accelerated the decline in plasma viraemia compared with ART alone, with statistically significant steeper slopes of decay in both antibody groups. The team also tracked cell-associated SIV DNA before and after depletion: control animals showed an average drop of 1.7 log units, CD4R1 recipients 2.7 log units, and bispecific antibody recipients 3.4 log units, a statistically significant advantage for the CCR5-targeting strategy. Notably, about ninety-five percent of proviruses detected during this early phase were intact, meaning the measured depletion applied to the replication-competent fraction of the reservoir rather than defective debris.

The immunological consequences of the two antibodies differed in instructive ways. CD4R1 produced profound and durable depletion of CD4-positive T cells and monocytes, with deficits persisting in some animals for twelve weeks. The bispecific antibody first caused a temporary CD3-positive lymphopenia that resolved within about four weeks, followed by a remarkably specific and long-lasting loss of CCR5-expressing cells. Circulating CCR5-positive memory CD4 and CD8 T cells were essentially eliminated, with three or fewer CCR5-positive CD4 memory cells per millilitre of blood detected for at least fourteen weeks after a single administration, and relative depletion persisting in some animals for thirty-four weeks or longer. Multiplex cytokine assays showed no significant inflammatory response attributable to the treatment, and the animals tolerated the procedure well, gaining weight comparably to controls, with only mild, transient hypersensitivity-type reactions in a few individuals.

The virological outcomes after ART withdrawal were striking. Every control animal rebounded within seven days, and six of eight CD4R1-treated animals rebounded within the same window. Among the seven bispecific antibody recipients, the first two rebounds did not occur until thirteen days after treatment interruption. Four others delayed rebound to ninety-seven or one hundred seventy-three days, or never rebounded at all during follow-up, which extended to twenty-nine weeks in one case and four years in another. A Cox proportional hazards model incorporating peak viral load and treatment group showed that both lower peak viraemia and bispecific antibody treatment independently delayed rebound, with the model estimating a ninety-five percent reduction in rebound hazard attributable to the bispecific antibody. Inheritance of protective MHC alleles did not predict outcome.

To determine whether the non-rebounding animals had truly cleared the virus, the researchers subjected three animals in durable remission to a battery of the most demanding tests available. High-sensitivity quantitative PCR with a detection limit of four copies per millilitre found no residual viraemia. The animals were then depleted of CD8-positive T cells with a rhesusized anti-CD8 antibody to remove any cytotoxic T lymphocyte control that might be suppressing hidden virus; rebound still did not occur, indicating that CD8 cells were not the mechanism of remission. Two of the animals were necropsied, and bone marrow, intestine, lung, spleen and multiple lymph nodes were examined by qPCR and by RNAscope and DNAscope in situ hybridization. No viral RNA or DNA was detected in any tissue, in sharp contrast to a viraemic control animal in which all six tissues tested were positive. Viral outgrowth assays from lymph-node cells failed to recover SIV in eight of eight attempts involving tens of millions of cells.

Mathematical modelling of the rebound data provided a quantitative estimate of how much the reservoir had shrunk. Using statistical tools that integrate viral-load and outgrowth information to infer the rate at which the remaining reservoir produces actively infected cells, the model estimated that CD4R1 treatment was associated with roughly a thirteenfold to twentyfold reduction in reservoir size, while the bispecific antibody was associated with a reduction of roughly 152-fold to 219-fold, after correcting for peak viral load and protective MHC alleles. These figures fall short of the two-thousand-fold reduction that modelling suggests is needed to delay rebound by a year in most people, which points to a second mechanism beyond simple cell killing. The authors argue that removing CCR5-positive cells also strips the virus of the targets it needs: reactivated virions encounter few infectable cells, nascent infectious foci cannot spread, and the residual reservoir decays through viral cytopathicity and cellular attrition. The case of the so-called Chicago patient, who achieved prolonged remission after a CCR5-Δ32 transplant despite a persistent reservoir, supports the idea that ART can be curative when infectable targets are scarce.

Several caveats temper the excitement. The intervention was delivered within days of infection, when the reservoir is small and still dominated by CCR5-positive cells; in chronic infection, the reservoir shifts toward memory and stem-cell-like CD4 subsets whose relationship to CCR5 is weaker, so the strategy may not translate directly to adults with long-standing HIV. The bispecific antibody also depletes CCR5-positive CD8 T cells and other uninfectable cells, raising questions about susceptibility to opportunistic infections, although lifelong CCR5 deficiency in Δ32 homozygotes is generally well tolerated apart from increased risk of symptomatic West Nile virus disease. It is also possible that part of the benefit comes from CCR5 blockade acting as an entry inhibitor rather than from depletion alone. Even so, the study demonstrates for the first time that a pharmacological intervention, a single shot, can achieve meaningful depletion of the replication-competent lentiviral reservoir and produce durable, apparently complete clearance in an animal model. For infants treated very soon after infection, and potentially as a component of combination immunotherapy for older patients, the result reopens a route to HIV cure that many in the field had considered closed.

Subject of Research: Depletion of the SIV reservoir in neonatal macaques using a CCR5/CD3 bispecific antibody

Article Title: SIV reservoir depletion and clearance from neonatal macaques following transient CCR5 depletion

Article References: Deere, J. D., Merriam, D., Machmach, K., Chang, W. L. W., Nekorchuk, M., Méndez-Lagares, G., Kieu, H., Dutra, J., Fontaine, J., Andal, D. R., Sipe, K., Heninger, E., Martin, M., Lu, W., Chin, N., Chen, C., Tran, B. C.-T., Salinas, J., Miller, C. N., … Hartigan-O’Connor, D. J. (2026). SIV reservoir depletion and clearance from neonatal macaques following transient CCR5 depletion. Nature Microbiology. https://doi.org/10.1038/s41564-026-02511-3

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02511-3

Keywords: HIV, SIV, viral reservoir, CCR5, bispecific antibody, macaques, antiretroviral therapy, latent infection, viral rebound, HIV cure, immunotherapy, neonatal infection

Diabetic retinopathy is a devastating complication of diabetes, characterized by progressive damage to the microvascular network that nourishes the light-sensitive tissue at the back of the eye. For decades, clinicians have focused their attention on the blood vessels themselves, treating the leaky capillaries and abnormal new vessel growth with laser therapy, injections of anti-VEGF drugs, or surgery. But a growing body of evidence points to a quieter culprit: chronic neuroinflammation, driven in large part by microglia, the resident immune cells of the retina. When these cells become chronically activated in a high-glucose environment, they release a cocktail of pro-inflammatory cytokines that erode the structural and functional integrity of the retinal vasculature from the outside in.

The research team approached the problem with an unusually systematic strategy. Rather than testing piceatannol blindly, they combined network pharmacology with transcriptomic analysis to map the compound’s potential molecular targets against the gene signatures of diabetic retinopathy. The computational screen identified 159 overlapping targets shared between the polyphenol and the disease. Among these, one receptor rose to the top: C-C chemokine receptor type 5, or CCR5, a G protein-coupled receptor best known to immunologists for its role in directing immune cell traffic and, famously, as an entry co-receptor exploited by HIV.

Molecular docking studies predicted that piceatannol could bind directly to CCR5, and the researchers went on to validate that binding affinity experimentally. This mattered because the receptor’s ligand, the chemokine CCL5, was significantly upregulated in the diabetic retina. The picture that emerged was of a signaling cascade, CCL5 binding to CCR5 and triggering the pro-inflammatory transcription factor NF-κB, that could potentially be intercepted at its receptor by a dietary polyphenol. Single-cell and expression analyses added a crucial layer of specificity: CCR5 was found to be expressed predominantly in retinal microglia, placing the immune cells squarely at the center of the pathway.

To test the compound’s effects, the team built disease models at two scales. In the laboratory, they exposed microglia to high glucose concentrations to mimic the diabetic metabolic environment. In living animals, they induced diabetes in mice using streptozotocin, a chemical that destroys insulin-producing cells and produces sustained hyperglycemia. The results in the animals were striking. Mice treated with piceatannol showed restored retinal thickness, reduced formation of acellular capillaries, the ghost vessels that mark dead capillary segments in diabetic retinas, and attenuated vascular leakage, the pathological seepage of fluid and proteins that ultimately destroys vision.

The cellular mechanism behind these improvements centered on microglial polarization. Microglia, like their peripheral cousins the macrophages, can adopt broadly two functional states. The M1 phenotype is pro-inflammatory, unleashing tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6, along with inducible nitric oxide synthase, all of which batter neighboring tissue. The M2 phenotype is restorative, promoting tissue repair through markers such as arginase-1, interleukin-10 and transforming growth factor beta 1. In the diabetic retina, the balance tips decisively toward M1. Piceatannol treatment, the researchers found, inhibited microglial activation and M1 polarization, reduced the secretion of pro-inflammatory cytokines, and promoted the expression of M2-associated markers, all through modulation of the CCL5/CCR5/NF-κB signaling pathway.

The downstream consequences for blood vessels were equally telling. In vitro experiments using human retinal endothelial cells showed that when high glucose pushed co-cultured microglia into their inflammatory state, the endothelial cells suffered: the tight junction proteins zonula occludens-1 and Claudin-5, which zip adjacent endothelial cells together to form the inner blood-retinal barrier, were degraded. Piceatannol suppressed the high-glucose-induced M1 polarization of the microglia and, as a result, preserved the expression of these tight junction proteins. In other words, the compound’s vascular protection appears to be indirect, mediated through the immune cells rather than through a direct effect on the endothelium itself.

The team then put the CCR5 hypothesis to a rigorous causal test. Using small interfering RNA to knock down CCR5 expression, and separately employing pharmacological activation of the receptor, they confirmed that CCR5 sits at the center of the protective mechanism. When the receptor was silenced, piceatannol’s benefits were diminished or recapitulated by the knockdown itself; when the pathway was artificially activated, the compound’s protective effects were counteracted. This pattern of gain- and loss-of-function evidence strengthens the argument that the CCL5/CCR5/NF-κB axis is not merely correlated with the disease process but is functionally required for the drug’s action.

The significance of the work lies partly in its translational framing. Piceatannol is a naturally occurring stilbenoid, meaning it is already present in the human food supply and has been studied for anti-inflammatory and vascular-protective properties in other contexts. Demonstrating a concrete, mechanistically defined role in diabetic retinopathy raises the possibility of repurposing a well-characterized molecule for a condition that currently affects tens of millions of people worldwide. The network pharmacology approach also offers a template for how natural products with dozens of potential targets can be systematically triaged, using docking, transcriptomics and weighted gene co-expression network analysis to converge on the handful of targets that actually matter.

Substantial caveats remain before patients should expect a piceatannol-based therapy. The findings come from cell culture and streptozotocin-induced mouse models, which reproduce key features of diabetic retinopathy but do not capture the full complexity of human disease, which unfolds over decades of metabolic stress. Questions about bioavailability, dosing, retinal drug delivery and long-term safety in a diabetic population remain open, and the study was funded by Chinese national and municipal science programs without reported involvement of commercial drug development. Still, by pinpointing a druggable receptor on retinal microglia and showing that a food-derived polyphenol can restrain the inflammatory cascade that dismantles the blood-retinal barrier, the study adds a compelling new chapter to the growing recognition that diabetic blindness is as much an immunological disease as a vascular one.

Subject of Research: The role of piceatannol in modulating microglial polarization via the CCL5/CCR5/NF-κB axis to protect retinal endothelial cells in diabetic retinopathy

Article Title: Piceatannol attenuates retinal endothelial cell dysfunction by modulating microglial polarization via the CCL5/CCR5/NF-κB axis

Article References: Tu, Y., Guo, Y., Zhang, Y., Sun, H., Zhu, X., Shen, G., Gu, Y., Xie, L., Song, E., & Zhu, M. (2026). Piceatannol attenuates retinal endothelial cell dysfunction by modulating microglial polarization via the CCL5/CCR5/NF-κB axis. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-08893-6

Image Credits: AI Generated

DOI: 10.1186/s12967-026-08893-6

Keywords: diabetic retinopathy, piceatannol, microglial polarization, CCR5, CCL5, NF-κB, retinal endothelial cells, blood-retinal barrier, neuroinflammation, tight junctions, network pharmacology, resveratrol