Berberine inhibits NLRP3 inflammasome activation and proinflammatory macrophage M1 polarization to accelerate peripheral nerve regeneration.

Berberine (BBR) has demonstrated potent anti-inflammatory effects by modulating macrophage polarization. Nevertheless, the precise mechanisms through which berberine regulates post-injury inflammation within the peripheral nerve system remain elusive. This study seeks to elucidate the role of BBR and its underlying mechanisms in inflammation following peripheral nerve injury (PNI). Adult male C57BL/6J mice subjected to PNI were administered daily doses of berberine (0, 60, 120, 180, 240 ​mg/kg) via gavage from day 1 through day 28. Evaluation of the sciatic function index (SFI) and paw withdrawal threshold revealed that BBR dose-dependently enhanced both motor and sensory functions. Immunofluorescent staining for anti-myelin basic protein (anti-MBP) and anti-neurofilament-200 (anti-NF-200), along with histological staining comprising hematoxylin-eosin (HE), luxol fast blue (LFB), and Masson staining, demonstrated that BBR dose-dependently promoted structural regeneration. Molecular analyses including qRT-PCR, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence confirmed that inactivation of the NLRP3 inflammasome by MCC950 shifted macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, while also impeding macrophage infiltration. Furthermore, BBR significantly downregulated the expression of the NLRP3 inflammasome and its associated molecules in macrophages, thereby mitigating NLRP3 inflammasome activation-induced macrophage M1 polarization and inflammation. In summary, BBR's neuroprotective effects were concomitant with the suppression of inflammation after PNI, achieved through the inhibition of NLRP3 inflammasome activation-induced macrophage M1 polarization.

Sun J ，Zeng Q ，Wu Z ，Huang L ，Sun T ，Ling C ，Zhang B ，Chen C ，Wang H ... -  《-》

Berberine inhibits palmitate-induced NLRP3 inflammasome activation by triggering autophagy in macrophages: A new mechanism linking berberine to insulin resistance improvement.

NLRP3 (Nod-like receptor family pyrin domain containing 3) inflammasome has been reported to contribute to obesity-induced inflammation and insulin resistance. However, there are few drugs targeting NLRP3 inflammasome for the treatment of insulin resistance. In the present study, we showed that berberine (BBR) significantly suppressed saturated fatty acid palmitate (PA)-induced NLRP3 inflammasome activation and interleukin-1β (IL-1β) release in macrophages, which was one of the most important mediators in the insulin sensitivity of adipose tissue. BBR treatment dramatically upregulated macrophage autophagic level while knockdown beclin1 and autophagy inhibitor reversed BBR's suppression on inflammasome. Furthermore, AMPK (adenosine monophosphate-activated protein kinase) inhibitor Adenine 9-β-d-arabinofuranoside (Ara-A) blocked most effects of BBR, suggesting that AMPK signals may be involved in BBR-induced macrophage autophagy. Importantly, BBR also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced insulin resistance model. Adoptive transfer of BBR-treated bone marrow-derived macrophages (BMDMs), which was induced by lipopolysaccharide (LPS) plus palmitate-bovine serum albumin (PA-BSA), significantly ameliorated insulin resistance of ob/ob mice as compared with control mice. However, the co-treatment of the BMDMs with autophagy inhibitor 3-Methyladenine (3-MA) reversed the effect of BBR almost completely. Taken together, BBR exerted its anti-inflammatory effects through activation of AMPK-dependent autophagy in adipose tissue macrophages (ATMs). This study amplified the mechanisms of BBR and its potential in attenuating insulin resistance.

Zhou H ，Feng L ，Xu F ，Sun Y ，Ma Y ，Zhang X ，Liu H ，Xu G ，Wu X ，Shen Y ，Sun Y ，Wu X ，Xu Q ... -  《-》

Down-regulation miR-146a-5p in Schwann cell-derived exosomes induced macrophage M1 polarization by impairing the inhibition on TRAF6/NF-κB pathway after peripheral nerve injury.

Both Schwann cell-derived exosomes (SC-Exos) and macrophagic sub-phenotypes are closely related to the regeneration and repair after peripheral nerve injury (PNI). However, the crosstalk between them is less clear. We aim to investigate the roles and underlying mechanisms of exosomes from normoxia-condition Schwann cell (Nor-SC-Exos) and from post-injury oxygen-glucose-deprivation-condition Schwann cell in regulating macrophagic sub-phenotypes and peripheral nerve injury repair. Both Nor-SC-Exos and OGD-SC-Exos were extracted through ultracentrifugation, identified by transmission electron microscopy (TEM), Nanosight tracking analysis (NTA) and western blotting. High-throughput sequencing was performed to explore the differential expression of microRNAs in both SC-Exos. In vitro, RAW264.7 macrophage was treated with two types of SC-Exos, M1 macrophagic markers (IL-10, Arg-1, TGF-β1) and M2 macrophagic markers (IL-6, IL-1β, TNF-α) were detected by enzyme-linked Immunosorbent Assay (ELISA) or qRT-PCR, and the expression of CD206, iNOS were detected via cellular immunofluorescence (IF) to judge macrophage sub-phenotypes. Dorsal root ganglion neurons (DRGns) were co-cultured with RAW264.7 cells treated with Nor-SC-Exos and OGD-SC-Exos, respectively, to explore their effect on neuron growth. In vivo, we established a sciatic nerve crush injury rat model. Nor-SC-Exos and OGD-SC-Exos were locally injected into the injury site. The mRNA expression of M1 macrophagic markers (IL-10, Arg-1, TGF-β1) and M2 macrophagic markers (IL-6, IL-1β, TNF-α) were detected by qRT-PCR to determine the sub-phenotype of macrophages in the injury site. IF was used to detect the expression of MBP and NF200, reflecting the myelin sheath and axon regeneration, and sciatic nerve function index (SFI) was measured to evaluate function repair. In vitro, Nor-SC-Exos promoted macrophage M2 polarization, increased anti-inflammation factors secretion, and facilitated axon elongation of DRGns. OGD-SC-Exos promoted M1 polarization, increased pro-inflammation factors secretion, and restrained axon elongation of DRGns. High-throughput sequencing and qRT-PCR results found that compared with Nor-SC-Exos, a shift from anti-inflammatory (pro-M2) to pro-inflammatory (pro-M1) of OGD-SC-Exos was closely related to the down-regulation of miR-146a-5p and its decreasing inhibition on TRAF6/NF-κB pathway after OGD injury. In vivo, we found Nor-SC-Exos and miR-146a-5p mimic promoted regeneration of myelin sheath and axon, and facilitated sciatic function repair via targeting TRAF6, while OGD-SC-Exos and miR-146a-5p inhibitor restrained them. Our study confirmed that miR-146a-5p was significantly decreased in SC-Exos under the ischemia-hypoxic microenvironment of the injury site after PNI, which mediated its shift from promoting macrophage M2 polarization (anti-inflammation) to promoting M1 polarization (pro-inflammation), thereby limiting axonal regeneration and functional recovery.

Sun J ，Liao Z ，Li Z ，Li H ，Wu Z ，Chen C ，Wang H ... -  《-》

Berberine suppresses influenza virus-triggered NLRP3 inflammasome activation in macrophages by inducing mitophagy and decreasing mitochondrial ROS.

Berberine (BBR) is an isoquinoline alkaloid extracted from several commonly used Chinese herbs. Our previous studies demonstrated BBR-mediated alleviation of lung injury due to inflammation and decrease in the mortality of mice with influenza viral pneumonia. The recent argument of autophagy against inflammatory responses has aroused wide concerns. This study focuses on the reactive oxygen species-Nod-like receptor protein 3 (ROS-NLRP3) pathway to investigate whether BBR inhibits NLRP3 inflammasome activation by inducing mitophagy. Our results demonstrate that BBR and mitochondrion-targeted superoxide dismutase mimetic (Mito-TEMPO; a specific mitochondrial ROS scavenger) significantly restricted NLRP3 inflammasome activation, increased mitochondrial membrane potential (MMP), and decreased mitochondrial ROS (mtROS) generation in J774A.1 macrophages infected with PR8 influenza virus. These observations suggest that the inhibitory effects of BBR on NLRP3 inflammasome activation were associated with the amelioration of mtROS generation. BBR treatment induced regular mitophagy, as evident from the increase in microtubule-associated protein 1 light chain 3 II, decrease in p62, colocalization of LC3 and mitochondria, and formation of autophagosomes. However, 3-methyladenine, an autophagy inhibitor, reversed the inhibitory effects of BBR on mitochondrial damage and NLRP3 inflammasome activation in influenza virus-infected macrophages, indicating the involvement of mitophagy in mediating the inhibitory effects of BBR on NLRP3 inflammasome activation. Furthermore, the knockdown of Bcl-2/adenovirus E18-19-kDa interacting protein 3 (BNIP3) expression attenuated the effects of BBR on mitophagy induction to some extent, suggesting that the BBR-induced mitophagy may be, at least in part, mediated in a BNIP3-dependent manner. Similar results were obtained in vivo using a mouse model of influenza viral pneumonia that was administered with BBR. Taken together, these findings suggest that restricting NLRP3 inflammasome activation by decreasing ROS generation through mitophagy induction may be crucial for the BBR-mediated alleviation of influenza virus-induced inflammatory lesions.

Liu H ，You L ，Wu J ，Zhao M ，Guo R ，Zhang H ，Su R ，Mao Q ，Deng D ，Hao Y ... -  《-》

IL-37 inhibits M1-like macrophage activation to ameliorate temporomandibular joint inflammation through the NLRP3 pathway.

IL-37 has been identified as an important anti-inflammatory and immunosuppressive factor. This study was undertaken to explore how IL-37 affects M1/M2-like macrophage polarization and thus contributes to anti-inflammatory processes in the temporomandibular joint. Western blotting, quantitative real-time PCR (qRT-PCR) and immunofluorescence were used to verify the IL-37-induced polarization shift from the M1 phenotype to the M2 phenotype, and the related key pathways were analysed by western blotting. Human chondrocytes were stimulated with M1-conditioned medium (CM) or IL-37-pretreated M1-CM, and inflammatory cytokines were detected. siRNA-IL-1R8 and MCC-950 were used to investigate the mechanism underlying the anti-inflammatory effects of IL-37. Complete Freund's adjuvant-induced and disc perforation-induced inflammation models were used for in vivo studies. Haematoxylin and eosin, immunohistochemical and safranin-O staining protocols were used to analyse histological changes in the synovium and condyle. Western blotting, qRT-PCR and immunofluorescence showed that IL-37 inhibited M1 marker expression and upregulated M2 marker expression. Western blotting and qRT-PCR showed that pretreatment with IL-37 suppressed inflammatory cytokine expression in chondrocytes. IL-37 inhibited the expression of NLRP3 and upregulated the expression of IL-1R8. Si-IL-1R8 and MCC-950 further confirmed that the anti-inflammatory properties of IL-37 were dependent on the presence of IL-1R8 and NLRP3. In vivo, IL-37 reduced synovial M1 marker expression and cartilage degeneration and increased M2 marker expression. IL-37 shifting of the polarization of macrophages from the pro-inflammatory M1 phenotype to the beneficial anti-inflammatory M2 phenotype seems to be a promising therapeutic strategy for treating temporomandibular joint inflammation.

Luo P ，Peng S ，Yan Y ，Ji P ，Xu J ... -  《-》