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To investigate the molecular mechanisms underlying early brain injury (EBI) after SAH, we established an in vivo SAH model in mice. The protein expression dynamics of <t>HSP60,</t> NLRP3, Caspase-1, and GSDMD in the ipsilateral cortex were analyzed at various time points using Western blot analysis. The results showed that compared with the sham-operated group, the expression levels of these proteins were significantly increased at 24 h after SAH (Fig. 2A, C, D, E). Further immunofluorescence co-localization analysis revealed a marked increase in the number of HSP60-positive cells in the brain tissue of the SAH group compared to the Sham group (Fig. 2B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

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1) Product Images from "HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage"

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

Journal: Inflammation

doi: 10.1007/s10753-025-02442-x

Figure Legend Snippet: To investigate the molecular mechanisms underlying early brain injury (EBI) after SAH, we established an in vivo SAH model in mice. The protein expression dynamics of HSP60, NLRP3, Caspase-1, and GSDMD in the ipsilateral cortex were analyzed at various time points using Western blot analysis. The results showed that compared with the sham-operated group, the expression levels of these proteins were significantly increased at 24 h after SAH (Fig. 2A, C, D, E). Further immunofluorescence co-localization analysis revealed a marked increase in the number of HSP60-positive cells in the brain tissue of the SAH group compared to the Sham group (Fig. 2B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: In Vivo, Expressing, Western Blot, Immunofluorescence

To simulate the pathological process following SAH at the cellular level and investigate its underlying molecular mechanisms, we established an in vitro SAH model by stimulating primary microglia with oxyhemoglobin (OxyHB). Western blot analysis revealed that, compared to the blank control group, the protein expression levels of HSP60, NLRP3, Caspase-1, and GSDMD were significantly upregulated after 24 h of OxyHB stimulation (Fig. 3A, C, D, E). Consistent with this, immunofluorescence co-localization results showed a pronounced increase in the number of HSP60-positive cells in the OxyHB-stimulated group compared to the blank control group (Fig. 3B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: To simulate the pathological process following SAH at the cellular level and investigate its underlying molecular mechanisms, we established an in vitro SAH model by stimulating primary microglia with oxyhemoglobin (OxyHB). Western blot analysis revealed that, compared to the blank control group, the protein expression levels of HSP60, NLRP3, Caspase-1, and GSDMD were significantly upregulated after 24 h of OxyHB stimulation (Fig. 3A, C, D, E). Consistent with this, immunofluorescence co-localization results showed a pronounced increase in the number of HSP60-positive cells in the OxyHB-stimulated group compared to the blank control group (Fig. 3B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: In Vitro, Western Blot, Control, Expressing, Immunofluorescence

To determine the optimal concentration of Mizoribine for inhibiting HSP60, Western blot was performed to assess the effect of different concentrations of Mizoribine on HSP60 expression. The results showed that the most significant inhibition of HSP60 was achieved at a dose of 100 mg/kg (Fig. 4A). Additionally, the CCK-8 assay revealed that Mizoribine concentration-dependently restored microglial viability under OxyHB stimulation (Fig. 4B). Further validation by Western blot confirmed that 80 µmol/L was the optimal concentration under in vitro conditions (Fig. 4C). Immunohistochemical results further indicated that Mizoribine treatment significantly reduced the abnormally elevated HSP60 expression in the SAH model (Fig. 4D). Furthermore, the efficacy of Mizoribine was further validated using another HSP60 inhibitor, Myrtucommulone A(Fig. 4E). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: To determine the optimal concentration of Mizoribine for inhibiting HSP60, Western blot was performed to assess the effect of different concentrations of Mizoribine on HSP60 expression. The results showed that the most significant inhibition of HSP60 was achieved at a dose of 100 mg/kg (Fig. 4A). Additionally, the CCK-8 assay revealed that Mizoribine concentration-dependently restored microglial viability under OxyHB stimulation (Fig. 4B). Further validation by Western blot confirmed that 80 µmol/L was the optimal concentration under in vitro conditions (Fig. 4C). Immunohistochemical results further indicated that Mizoribine treatment significantly reduced the abnormally elevated HSP60 expression in the SAH model (Fig. 4D). Furthermore, the efficacy of Mizoribine was further validated using another HSP60 inhibitor, Myrtucommulone A(Fig. 4E). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: Concentration Assay, Western Blot, Expressing, Inhibition, CCK-8 Assay, Biomarker Discovery, In Vitro, Immunohistochemical staining

To investigate the role of HSP60 in microglial pyroptosis after SAH, the expression of key proteins in the NLRP3 inflammasome pathway, including NLRP3, pro-Caspase-1/Cleaved Caspase-1, and ASC, was detected by Western blot. In vivo results showed that inhibition of HSP60 with Mizoribine significantly reduced the expression levels of these proteins (Fig. 5A, E, I). A consistent trend was observed in cellular experiments (Fig. 5C, G, K). For further verification, immunofluorescence double staining was performed to label microglia and related target proteins in both in vivo and in vitro models. The changes in protein expression were consistent with the Western blot results (Fig. 5B, F, J, D, H, L). In summary, Mizoribine treatment significantly suppressed the activation of the NLRP3 inflammasome after SAH, indicating that HSP60 is involved in regulating SAH-induced microglial pyroptosis. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: To investigate the role of HSP60 in microglial pyroptosis after SAH, the expression of key proteins in the NLRP3 inflammasome pathway, including NLRP3, pro-Caspase-1/Cleaved Caspase-1, and ASC, was detected by Western blot. In vivo results showed that inhibition of HSP60 with Mizoribine significantly reduced the expression levels of these proteins (Fig. 5A, E, I). A consistent trend was observed in cellular experiments (Fig. 5C, G, K). For further verification, immunofluorescence double staining was performed to label microglia and related target proteins in both in vivo and in vitro models. The changes in protein expression were consistent with the Western blot results (Fig. 5B, F, J, D, H, L). In summary, Mizoribine treatment significantly suppressed the activation of the NLRP3 inflammasome after SAH, indicating that HSP60 is involved in regulating SAH-induced microglial pyroptosis. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: Expressing, Western Blot, In Vivo, Inhibition, Immunofluorescence, Double Staining, In Vitro, Activation Assay

To further explore the mechanism of HSP60 in microglial pyroptosis, the expression of GSDMD-N, the key cleaved and activated form of GSDMD that mediates plasma membrane pore formation, was detected. Western blot analysis showed that the protein levels of GSDMD-F/GSDMD-N were significantly reduced in the Mizoribine treatment group compared with the SAH group, a finding consistently validated in both in vivo and in vitro experiments (Fig. 6A, C). Further co-localization analysis of microglia via immunofluorescence revealed a marked reduction in positive signals for the target proteins in the Mizoribine group (Fig. 6B, D). Transmission electron microscopy observations indicated that Mizoribine treatment significantly ameliorated ultrastructural alterations associated with pyroptosis, including plasma membrane pore formation (red arrows) (Fig. 6E). Additionally, assessment of plasma membrane integrity in primary microglia by immunofluorescence staining demonstrated that inhibition of HSP60 markedly alleviated SAH-induced membrane integrity damage (Fig. 6F). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: To further explore the mechanism of HSP60 in microglial pyroptosis, the expression of GSDMD-N, the key cleaved and activated form of GSDMD that mediates plasma membrane pore formation, was detected. Western blot analysis showed that the protein levels of GSDMD-F/GSDMD-N were significantly reduced in the Mizoribine treatment group compared with the SAH group, a finding consistently validated in both in vivo and in vitro experiments (Fig. 6A, C). Further co-localization analysis of microglia via immunofluorescence revealed a marked reduction in positive signals for the target proteins in the Mizoribine group (Fig. 6B, D). Transmission electron microscopy observations indicated that Mizoribine treatment significantly ameliorated ultrastructural alterations associated with pyroptosis, including plasma membrane pore formation (red arrows) (Fig. 6E). Additionally, assessment of plasma membrane integrity in primary microglia by immunofluorescence staining demonstrated that inhibition of HSP60 markedly alleviated SAH-induced membrane integrity damage (Fig. 6F). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: Expressing, Clinical Proteomics, Membrane, Western Blot, In Vivo, In Vitro, Immunofluorescence, Transmission Assay, Electron Microscopy, Staining, Inhibition

The expression levels of inflammatory cytokines following microglial pyroptosis were detected by Western blot. The results showed that the expression of IL-1β and its cleaved form, Cleaved IL-1β, was significantly increased after SAH, while Mizoribine treatment effectively inhibited the expression of both proteins (Fig. 7A, B). Further evaluation of the concentrations of IL-18 and IL-1β by ELISA revealed that Mizoribine significantly reduced the SAH-induced elevation of both cytokines (Fig. 7C–F). Moreover, immunofluorescence detection of microglial activation showed that inhibition of HSP60 reduced the number of M1-positive microglia and promoted an increase in M2-type microglia (Fig. 7G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: The expression levels of inflammatory cytokines following microglial pyroptosis were detected by Western blot. The results showed that the expression of IL-1β and its cleaved form, Cleaved IL-1β, was significantly increased after SAH, while Mizoribine treatment effectively inhibited the expression of both proteins (Fig. 7A, B). Further evaluation of the concentrations of IL-18 and IL-1β by ELISA revealed that Mizoribine significantly reduced the SAH-induced elevation of both cytokines (Fig. 7C–F). Moreover, immunofluorescence detection of microglial activation showed that inhibition of HSP60 reduced the number of M1-positive microglia and promoted an increase in M2-type microglia (Fig. 7G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, ** P < 0.01, *** P < 0.001

Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Activation Assay, Inhibition

Western blot analysis of the expression levels of blood-brain barrier tight junction proteins ZO-1 and Occludin showed a significant decrease after SAH, while Mizoribine intervention markedly alleviated the SAH-induced downregulation of these proteins (Fig. 8A, B). Measurement of brain water content indicated that Mizoribine treatment effectively reduced cerebral edema after SAH (Fig. 8C). Neurological function assessments, including the modified Garcia score, beam walking test, and water maze test, demonstrated that mice treated with the HSP60 inhibitor Mizoribine achieved higher scores in all behavioral tests, indicating significant improvement in neurological deficits (Fig. 8D–F). Further evaluation of neuronal damage by TUNEL staining and Nissl staining revealed that inhibition of HSP60 significantly reduced neuronal apoptosis and improved Nissl body integrity after SAH, suggesting a neuroprotective effect of Mizoribine treatment (Fig. 8G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. In addition, two-way repeated measures ANOVA were applied to analyze the data of long-term neurological functions. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01

Figure Legend Snippet: Western blot analysis of the expression levels of blood-brain barrier tight junction proteins ZO-1 and Occludin showed a significant decrease after SAH, while Mizoribine intervention markedly alleviated the SAH-induced downregulation of these proteins (Fig. 8A, B). Measurement of brain water content indicated that Mizoribine treatment effectively reduced cerebral edema after SAH (Fig. 8C). Neurological function assessments, including the modified Garcia score, beam walking test, and water maze test, demonstrated that mice treated with the HSP60 inhibitor Mizoribine achieved higher scores in all behavioral tests, indicating significant improvement in neurological deficits (Fig. 8D–F). Further evaluation of neuronal damage by TUNEL staining and Nissl staining revealed that inhibition of HSP60 significantly reduced neuronal apoptosis and improved Nissl body integrity after SAH, suggesting a neuroprotective effect of Mizoribine treatment (Fig. 8G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. In addition, two-way repeated measures ANOVA were applied to analyze the data of long-term neurological functions. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01

Techniques Used: Western Blot, Expressing, Modification, TUNEL Assay, Staining, Inhibition

$To elucidate the central role of HSP60 in neuroinflammation after SAH, we first confirmed a direct protein-protein interaction between HSP60 and TLR4 by co-immunoprecipitation (Fig. 9A). Building on this interaction, we further validated in vivo through intrathecal injection of recombinant HSP60 protein that it can induce neuroinflammation by activating the TLR4 and its downstream MyD88/NF-κB signaling pathway (Fig. 9B, C, D). To evaluate the therapeutic potential of targeting this pathway, nuclear-cytoplasmic fractionation was performed. The results showed that the HSP60 inhibitor Mizoribine effectively reduced the nuclear accumulation level of phosphorylated NF-κB (p-p65) after SAH (Fig. 9E), mechanistically confirming that HSP60 inhibition blocks the activation of the downstream NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001$
Figure Legend Snippet: To elucidate the central role of HSP60 in neuroinflammation after SAH, we first confirmed a direct protein-protein interaction between HSP60 and TLR4 by co-immunoprecipitation (Fig. 9A). Building on this interaction, we further validated in vivo through intrathecal injection of recombinant HSP60 protein that it can induce neuroinflammation by activating the TLR4 and its downstream MyD88/NF-κB signaling pathway (Fig. 9B, C, D). To evaluate the therapeutic potential of targeting this pathway, nuclear-cytoplasmic fractionation was performed. The results showed that the HSP60 inhibitor Mizoribine effectively reduced the nuclear accumulation level of phosphorylated NF-κB (p-p65) after SAH (Fig. 9E), mechanistically confirming that HSP60 inhibition blocks the activation of the downstream NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: Immunoprecipitation, In Vivo, Injection, Recombinant, Fractionation, Inhibition, Activation Assay

Western blot analysis of the expression levels of key pathway proteins, including TLR4, MyD88, and p-NF-κB, in SAH mice showed a significant increase after SAH, which was markedly reversed by Mizoribine-mediated inhibition of HSP60 (Fig. 10A, E, I). Consistent results were obtained in experiments stimulating microglia with OxyHB (Fig. 10C, G, K). Further co-localization analysis of microglia and the respective pathway proteins by immunofluorescence (Fig. 10B, F, J) demonstrated that Mizoribine treatment significantly attenuated SAH-induced activation of TLR4, MyD88, and p-NF-κB in microglia. Identical results were observed in in vitro experiments (Fig. 10D, H, L). These findings collectively indicate that HSP60 likely participates in the process of microglial pyroptosis after SAH by regulating the TLR4/MyD88/NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure Legend Snippet: Western blot analysis of the expression levels of key pathway proteins, including TLR4, MyD88, and p-NF-κB, in SAH mice showed a significant increase after SAH, which was markedly reversed by Mizoribine-mediated inhibition of HSP60 (Fig. 10A, E, I). Consistent results were obtained in experiments stimulating microglia with OxyHB (Fig. 10C, G, K). Further co-localization analysis of microglia and the respective pathway proteins by immunofluorescence (Fig. 10B, F, J) demonstrated that Mizoribine treatment significantly attenuated SAH-induced activation of TLR4, MyD88, and p-NF-κB in microglia. Identical results were observed in in vitro experiments (Fig. 10D, H, L). These findings collectively indicate that HSP60 likely participates in the process of microglial pyroptosis after SAH by regulating the TLR4/MyD88/NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques Used: Western Blot, Expressing, Inhibition, Immunofluorescence, Activation Assay, In Vitro

During early brain injury after subarachnoid hemorrhage (SAH), HSP60 expression is significantly upregulated. HSP60 activates the TLR4/MyD88/NF-κB signaling pathway in microglia, inducing upregulation of NLRP3 expression and accumulation of inflammatory cytokine precursors such as pro-IL-1β and pro-IL-18. Elevated NLRP3 further promotes the assembly of the NLRP3 inflammasome, which activates Caspase-1 by cleaving its precursor pro-Caspase-1 into the enzymatically active form Cleaved Caspase-1. Activated Caspase-1 cleaves these cytokine precursors into their mature and biologically active forms, IL-1β and IL-18, and also cleaves gasdermin D (GSDMD), generating its N-terminal fragment (GSDMD-N). GSDMD-N forms pores in the cell membrane, mediating the massive release of mature inflammatory cytokines, ultimately triggering and exacerbating microglial pyroptosis, thereby significantly aggravating the process of early brain injury after SAH. Inhibiting HSP60 with Mizoribine can significantly alleviate HSP60-induced early brain injury after SAH

Figure Legend Snippet: During early brain injury after subarachnoid hemorrhage (SAH), HSP60 expression is significantly upregulated. HSP60 activates the TLR4/MyD88/NF-κB signaling pathway in microglia, inducing upregulation of NLRP3 expression and accumulation of inflammatory cytokine precursors such as pro-IL-1β and pro-IL-18. Elevated NLRP3 further promotes the assembly of the NLRP3 inflammasome, which activates Caspase-1 by cleaving its precursor pro-Caspase-1 into the enzymatically active form Cleaved Caspase-1. Activated Caspase-1 cleaves these cytokine precursors into their mature and biologically active forms, IL-1β and IL-18, and also cleaves gasdermin D (GSDMD), generating its N-terminal fragment (GSDMD-N). GSDMD-N forms pores in the cell membrane, mediating the massive release of mature inflammatory cytokines, ultimately triggering and exacerbating microglial pyroptosis, thereby significantly aggravating the process of early brain injury after SAH. Inhibiting HSP60 with Mizoribine can significantly alleviate HSP60-induced early brain injury after SAH

Techniques Used: Expressing, Membrane

Image Search Results

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To investigate the molecular mechanisms underlying early brain injury (EBI) after SAH, we established an in vivo SAH model in mice. The protein expression dynamics of HSP60, NLRP3, Caspase-1, and GSDMD in the ipsilateral cortex were analyzed at various time points using Western blot analysis. The results showed that compared with the sham-operated group, the expression levels of these proteins were significantly increased at 24 h after SAH (Fig. 2A, C, D, E). Further immunofluorescence co-localization analysis revealed a marked increase in the number of HSP60-positive cells in the brain tissue of the SAH group compared to the Sham group (Fig. 2B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Intrathecal injection of 40 μg HSP60 (MCE, # HY-P73916A, China) can specifically activate the TLR4-MyD88 pathway, and was used to establish a model of neuroinflammatory brain injury [ ].

Techniques: In Vivo, Expressing, Western Blot, Immunofluorescence

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To simulate the pathological process following SAH at the cellular level and investigate its underlying molecular mechanisms, we established an in vitro SAH model by stimulating primary microglia with oxyhemoglobin (OxyHB). Western blot analysis revealed that, compared to the blank control group, the protein expression levels of HSP60, NLRP3, Caspase-1, and GSDMD were significantly upregulated after 24 h of OxyHB stimulation (Fig. 3A, C, D, E). Consistent with this, immunofluorescence co-localization results showed a pronounced increase in the number of HSP60-positive cells in the OxyHB-stimulated group compared to the blank control group (Fig. 3B). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: In Vitro, Western Blot, Control, Expressing, Immunofluorescence

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To determine the optimal concentration of Mizoribine for inhibiting HSP60, Western blot was performed to assess the effect of different concentrations of Mizoribine on HSP60 expression. The results showed that the most significant inhibition of HSP60 was achieved at a dose of 100 mg/kg (Fig. 4A). Additionally, the CCK-8 assay revealed that Mizoribine concentration-dependently restored microglial viability under OxyHB stimulation (Fig. 4B). Further validation by Western blot confirmed that 80 µmol/L was the optimal concentration under in vitro conditions (Fig. 4C). Immunohistochemical results further indicated that Mizoribine treatment significantly reduced the abnormally elevated HSP60 expression in the SAH model (Fig. 4D). Furthermore, the efficacy of Mizoribine was further validated using another HSP60 inhibitor, Myrtucommulone A(Fig. 4E). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Concentration Assay, Western Blot, Expressing, Inhibition, CCK-8 Assay, Biomarker Discovery, In Vitro, Immunohistochemical staining

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To investigate the role of HSP60 in microglial pyroptosis after SAH, the expression of key proteins in the NLRP3 inflammasome pathway, including NLRP3, pro-Caspase-1/Cleaved Caspase-1, and ASC, was detected by Western blot. In vivo results showed that inhibition of HSP60 with Mizoribine significantly reduced the expression levels of these proteins (Fig. 5A, E, I). A consistent trend was observed in cellular experiments (Fig. 5C, G, K). For further verification, immunofluorescence double staining was performed to label microglia and related target proteins in both in vivo and in vitro models. The changes in protein expression were consistent with the Western blot results (Fig. 5B, F, J, D, H, L). In summary, Mizoribine treatment significantly suppressed the activation of the NLRP3 inflammasome after SAH, indicating that HSP60 is involved in regulating SAH-induced microglial pyroptosis. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Expressing, Western Blot, In Vivo, Inhibition, Immunofluorescence, Double Staining, In Vitro, Activation Assay

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To further explore the mechanism of HSP60 in microglial pyroptosis, the expression of GSDMD-N, the key cleaved and activated form of GSDMD that mediates plasma membrane pore formation, was detected. Western blot analysis showed that the protein levels of GSDMD-F/GSDMD-N were significantly reduced in the Mizoribine treatment group compared with the SAH group, a finding consistently validated in both in vivo and in vitro experiments (Fig. 6A, C). Further co-localization analysis of microglia via immunofluorescence revealed a marked reduction in positive signals for the target proteins in the Mizoribine group (Fig. 6B, D). Transmission electron microscopy observations indicated that Mizoribine treatment significantly ameliorated ultrastructural alterations associated with pyroptosis, including plasma membrane pore formation (red arrows) (Fig. 6E). Additionally, assessment of plasma membrane integrity in primary microglia by immunofluorescence staining demonstrated that inhibition of HSP60 markedly alleviated SAH-induced membrane integrity damage (Fig. 6F). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot, In Vivo, In Vitro, Immunofluorescence, Transmission Assay, Electron Microscopy, Staining, Inhibition

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: The expression levels of inflammatory cytokines following microglial pyroptosis were detected by Western blot. The results showed that the expression of IL-1β and its cleaved form, Cleaved IL-1β, was significantly increased after SAH, while Mizoribine treatment effectively inhibited the expression of both proteins (Fig. 7A, B). Further evaluation of the concentrations of IL-18 and IL-1β by ELISA revealed that Mizoribine significantly reduced the SAH-induced elevation of both cytokines (Fig. 7C–F). Moreover, immunofluorescence detection of microglial activation showed that inhibition of HSP60 reduced the number of M1-positive microglia and promoted an increase in M2-type microglia (Fig. 7G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, ** P < 0.01, *** P < 0.001

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Activation Assay, Inhibition

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: Western blot analysis of the expression levels of blood-brain barrier tight junction proteins ZO-1 and Occludin showed a significant decrease after SAH, while Mizoribine intervention markedly alleviated the SAH-induced downregulation of these proteins (Fig. 8A, B). Measurement of brain water content indicated that Mizoribine treatment effectively reduced cerebral edema after SAH (Fig. 8C). Neurological function assessments, including the modified Garcia score, beam walking test, and water maze test, demonstrated that mice treated with the HSP60 inhibitor Mizoribine achieved higher scores in all behavioral tests, indicating significant improvement in neurological deficits (Fig. 8D–F). Further evaluation of neuronal damage by TUNEL staining and Nissl staining revealed that inhibition of HSP60 significantly reduced neuronal apoptosis and improved Nissl body integrity after SAH, suggesting a neuroprotective effect of Mizoribine treatment (Fig. 8G, H). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. In addition, two-way repeated measures ANOVA were applied to analyze the data of long-term neurological functions. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01

Techniques: Western Blot, Expressing, Modification, TUNEL Assay, Staining, Inhibition

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: To elucidate the central role of HSP60 in neuroinflammation after SAH, we first confirmed a direct protein-protein interaction between HSP60 and TLR4 by co-immunoprecipitation (Fig. 9A). Building on this interaction, we further validated in vivo through intrathecal injection of recombinant HSP60 protein that it can induce neuroinflammation by activating the TLR4 and its downstream MyD88/NF-κB signaling pathway (Fig. 9B, C, D). To evaluate the therapeutic potential of targeting this pathway, nuclear-cytoplasmic fractionation was performed. The results showed that the HSP60 inhibitor Mizoribine effectively reduced the nuclear accumulation level of phosphorylated NF-κB (p-p65) after SAH (Fig. 9E), mechanistically confirming that HSP60 inhibition blocks the activation of the downstream NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Immunoprecipitation, In Vivo, Injection, Recombinant, Fractionation, Inhibition, Activation Assay

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: Western blot analysis of the expression levels of key pathway proteins, including TLR4, MyD88, and p-NF-κB, in SAH mice showed a significant increase after SAH, which was markedly reversed by Mizoribine-mediated inhibition of HSP60 (Fig. 10A, E, I). Consistent results were obtained in experiments stimulating microglia with OxyHB (Fig. 10C, G, K). Further co-localization analysis of microglia and the respective pathway proteins by immunofluorescence (Fig. 10B, F, J) demonstrated that Mizoribine treatment significantly attenuated SAH-induced activation of TLR4, MyD88, and p-NF-κB in microglia. Identical results were observed in in vitro experiments (Fig. 10D, H, L). These findings collectively indicate that HSP60 likely participates in the process of microglial pyroptosis after SAH by regulating the TLR4/MyD88/NF-κB signaling pathway. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for comparisons among multiple groups. Data are presented as mean ± SD; ns indicates not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Techniques: Western Blot, Expressing, Inhibition, Immunofluorescence, Activation Assay, In Vitro

Journal: Inflammation

Article Title: HSP60 Mediates NLRP3 Inflammasome-Dependent Microglial Pyroptosis Via the TLR4/MyD88/NF-κB Signaling Axis After Subarachnoid Hemorrhage

doi: 10.1007/s10753-025-02442-x

Figure Lengend Snippet: During early brain injury after subarachnoid hemorrhage (SAH), HSP60 expression is significantly upregulated. HSP60 activates the TLR4/MyD88/NF-κB signaling pathway in microglia, inducing upregulation of NLRP3 expression and accumulation of inflammatory cytokine precursors such as pro-IL-1β and pro-IL-18. Elevated NLRP3 further promotes the assembly of the NLRP3 inflammasome, which activates Caspase-1 by cleaving its precursor pro-Caspase-1 into the enzymatically active form Cleaved Caspase-1. Activated Caspase-1 cleaves these cytokine precursors into their mature and biologically active forms, IL-1β and IL-18, and also cleaves gasdermin D (GSDMD), generating its N-terminal fragment (GSDMD-N). GSDMD-N forms pores in the cell membrane, mediating the massive release of mature inflammatory cytokines, ultimately triggering and exacerbating microglial pyroptosis, thereby significantly aggravating the process of early brain injury after SAH. Inhibiting HSP60 with Mizoribine can significantly alleviate HSP60-induced early brain injury after SAH

Techniques: Expressing, Membrane

HSP90AA1 plays a pivotal role in the heat stress response of liver cancer cells. (a) WB analysis was employed to assess alterations in the expression levels of HSP110, HSP90AA1, HSP70, and HSP60 in liver cancer cells following exposure to heat stress. (b) To evaluate transfection efficiency, WB analysis was performed to assess the effects of HSP110 or HSP90AA1 silence in HepG2 and Huh7 cells. (c) MTT assay was used to evaluate changes in cell viability of HepG2 and Huh7 cells after heat stress treatment with or without the silencing of HSP90 and HSP110, respectively. (d) Flow cytometry was used to analyze changes in the apoptosis rate of HepG2 and Huh7 cells after heat stress treatment with or without the silencing of HSP90 and HSP110, respectively. All experiments were performed with at least three replicates. Statistical significance was denoted as * P < 0.05 and ** P < 0.01.

Journal: Cell Stress & Chaperones

Article Title: Heat stress-induced heat shock protein 90 alpha family class A member 1 upregulation stabilizes yes-associated protein through ubiquitination inhibition to boost hepatic cancer radiofrequency hyperthermia resistance

doi: 10.1016/j.cstres.2025.100140

Figure Lengend Snippet: HSP90AA1 plays a pivotal role in the heat stress response of liver cancer cells. (a) WB analysis was employed to assess alterations in the expression levels of HSP110, HSP90AA1, HSP70, and HSP60 in liver cancer cells following exposure to heat stress. (b) To evaluate transfection efficiency, WB analysis was performed to assess the effects of HSP110 or HSP90AA1 silence in HepG2 and Huh7 cells. (c) MTT assay was used to evaluate changes in cell viability of HepG2 and Huh7 cells after heat stress treatment with or without the silencing of HSP90 and HSP110, respectively. (d) Flow cytometry was used to analyze changes in the apoptosis rate of HepG2 and Huh7 cells after heat stress treatment with or without the silencing of HSP90 and HSP110, respectively. All experiments were performed with at least three replicates. Statistical significance was denoted as * P < 0.05 and ** P < 0.01.

Article Snippet: HSP60 , Proteintech , 15282-1-AP , 1:2000.

Techniques: Expressing, Transfection, MTT Assay, Flow Cytometry

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