Design, synthesis and biological evaluation of matrine contains benzimidazole derivatives as dual TOPOI and PARP inhibitors for cancer therapy.

TOPOI inhibitors have long been a focal point in the research and development of antitumor drugs. PARP-1 plays a crucial role in repairing DNA damage induced by TOPOI inhibitors. Thus, concurrent inhibition of TOPOI and PARP-1 has the potential to augment drug activity. Matrine, characterized by low toxicity and good water solubility, offers advantageous properties. In this investigation, a series of benzimidazole matrine derivatives were designed and synthesized using matrine as the lead compound with the aim of developing dual inhibitors targeting both TOPOI and PARP-1. Among these derivatives, Compound B6 exhibited potent inhibitory effects on PARP-1 and TOPOI, effectively suppressing cancer cell proliferation and migration. Mechanistic assessments revealed that B6 induced DNA damage in HGC-27 cells, leading to G0/G1 cell cycle arrest and significant apoptosis. Molecular docking experiments demonstrated that B6 can effectively enter the active pocket of target proteins, where it forms stable hydrogen bonds with amino acid residues. In vivo, experiments demonstrated that B6 exhibited antitumor activity comparable to that of the positive control drug. The tumor growth inhibition rates (TGIs) for irinotecan, B6 and matrine were 87.0%, 75.4% and 9.7%, respectively. Importantly, B6 demonstrated lower toxicity than the positive control drug. Our findings suggest that TOPOI and PARP-1 may represent potential targets for matrine and B6 emerges as a promising candidate for cancer therapy.

Inhibition of the interaction between microglial adenosine 2A receptor and NLRP3 inflammasome attenuates neuroinflammation posttraumatic brain injury.

AIMS: Adenosine 2A receptor (A2A R) is widely expressed in the brain and plays important roles in neuroinflammation, and the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system while the regulation of A2A R on it in the central nervous system (CNS) has not been clarified. METHODS: The effects of microglial A2A R on NLRP3 inflammasome assembly and activation were investigated in wild-type, A2A R- or NLRP3-knockout primary microglia with pharmacological treatment. Microglial A2A R or NLRP3 conditional knockout mice were used to interrogate the effects of this regulation on neuroinflammation posttraumatic brain injury (TBI). RESULTS: We found that A2A R directly interacted with NLRP3 and facilitated NLRP3 inflammasome assembly and activation in primary microglia while having no effects on mRNA levels of inflammasome components. Inhibition of the interaction via A2A R agonist or knockout attenuated inflammasome assembly and activation in vitro. In the TBI model, microglial A2A R and NLRP3 were co-expressed at high levels in microglia next to the peri-injured cortex, and abrogating of this interaction by microglial NLRP3 or A2A R conditional knockout attenuated the neurological deficits and neuropathology post-TBI via reducing the NLRP3 inflammasome activation. CONCLUSION: Our results demonstrated that inhibition of the interaction between A2A R and NLRP3 in microglia could mitigate the NLRP3 inflammasome assembly and activation and ameliorate the neuroinflammation post-TBI. It provides new insights into the effects of A2A R on neuroinflammation regulation post-TBI and offers a potential target for the treatment of NLRP3 inflammasome-related CNS diseases.

HET0016 inhibits neuronal pyroptosis in the immature brain post-TBI via the p38 MAPK signaling pathway.

Traumatic brain injury (TBI) is a serious health threat worldwide, especially for the younger demographic. Our previous study demonstrated that HET0016 (a specific inhibitor of 20-hydroxyeicosatetraenoic acid synthesis) can decrease the lesion volume in the immature brain post-TBI; however, its mechanism of action and its association with pyroptosis post-TBI are unclear. In this study, we established a controlled cortical impact (CCI) injury rat model (postnatal day 9-10) and observed that increased expression of indicators for pyroptosis, including NLR family pyrin domain containing 3 (NLRP3), caspase-1 and gasdermin D (GSDMD) proteins and interleukin (IL)-18/IL-1ß mRNA during the acute phase of TBI, especially on post-injury day (PID) 1. Additionally, we found that caspase-1 was primarily expressed in the neurons and microglia. HET0016 (1 mg/kg/d, ip, 3 consecutive days since TBI) reduced the lesion volume; neuronal death; expression of NLRP3, caspase-1, and GSDMD; and expression of IL-18/IL-1ß mRNA. Bioinformatics analysis suggested involvement of mitogen-activated protein kinase (MAPK) signaling pathway in the HET0016-mediated neuroprotective role against TBI in the immature brain. Western blot analysis revealed reduced expression of p-p38 MAPK and nuclear factor-kappa B (NF-κB) p65 in the neurons and microglia upon HET0016 treatment in TBI rats. In cultured primary cortical neurons subjected to oxygen-glucose deprivation/re-oxygenation (OGD) + (lipopolysaccharide) LPS, HET0016-induced the reduction of p-p38 MAPK, NLRP3, cleaved-caspase-1, GSDMD, IL-18, and IL-1ß was reversed by co-treatment with p38 MAPK activator as well as NLRP3 agonist. Therefore, we conclude that pyroptosis is involved in neuronal death in the immature brains post-TBI and that HET0016 administration can alleviate neuronal pyroptosis possibly via inhibiting the phosphorylation of p38 MAPK.

Ablation of GSDMD Attenuates Neurological Deficits and Neuropathological Alterations After Traumatic Brain Injury.

Pyroptosis plays a significant role in neuroinflammation after traumatic brain injury (TBI). However, the role of pyroptosis executor Gasdermin D (GSDMD) in neurological deficits and neuropathological alterations after TBI have not been elucidated. Our results demonstrated that GSDMD-KO exerted striking neuroprotective effects on motor dysfunction and neuropathological alterations (loss of synaptic proteins, microglia activation, astrogliosis, dendrite injury, and neuron death) at 3 days after TBI. GSDMD-KO inhibited the expression and release of pro-inflammatory cytokine releases (IL-1ß and TNF-α) while promoting those of anti-inflammatory cytokines (IL-10 and TGF-ß1). The temporal pattern of diverse inflammasome signals showed long-lasting elevations of NLRP3, caspase 1, and caspase 1 p20 after TBI, rather than NLRP1, NLRC4 or AIM2, similar to the change in GSDMD postinjury; and NLRP3-KO not only inhibited the expression and cleavage of GSDMD but also attenuated the loss of synaptic proteins and neurological deficits. Notably, RNA sequencing showed both GSDMD-KO and NLRP3-KO reversed the global expression of neuroinflammation- and neuropathology-related genes after TBI. Our findings proved that the inhibition of GSDMD exerts neuroprotective effects after TBI and is mainly driven by the NLRP3 inflammasome. GSDMD serves as a potent therapeutic target for the treatment of TBI.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.