Hematoporphyrin monomethyl ether-mediated photodynamic therapy selectively kills sarcomas by inducing apoptosis.

We investigated the antitumor effect and mechanism of hematoporphyrin monomethyl ether-mediated photodynamic therapy (HMME-PDT) in sarcomas. Intracellular uptake of HMME by osteosarcoma cells (LM8 and K7) was time- and dose-dependent, while this was not observed for myoblast cells (C2C12) and fibroblast cells (NIH/3T3). HMME-PDT markedly inhibited the proliferation of sarcoma cell lines (LM8, MG63, Saos-2, SW1353, TC71, and RD) (P<0.05), and the killing effect was improved with increased HMME concentration and energy intensity. Flow cytometry analysis revealed that LM8, MG63, and Saos-2 cells underwent apoptosis after treatment with HMME-PDT. Additionally, apoptosis was induced after HMME-PDT in a three-dimensional culture of osteosarcoma cells. Hoechst 33342 staining confirmed apoptosis. Cell death caused by PDT was rescued by an irreversible inhibitor (Z-VAD-FMK) of caspase. However, cell viability was not markedly decreased compared with the HMME-PDT group. Expression levels of caspase-1, caspase-3, caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP) proteins were markedly up-regulated in the treatment groups and increased with HMME concentration as determined by western blot analysis. In vivo, tumor volume markedly decreased at 7-16 days post-PDT. Hematoxylin and eosin staining revealed widespread necrotic and infiltrative inflammatory cells in the HMME-PDT group. Immunohistochemistry analysis also showed that caspase-1, caspase-3, caspase-6, caspase-9, and PARP proteins were significantly increased in the HMME-PDT group. These results indicate that HMME-PDT has a potent killing effect on osteosarcoma cells in vitro and significantly inhibits tumor growth in vivo, which is associated with the caspase-dependent pathway

Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

AbstractRenal ischemia-reperfusion (I/R) injury leads to irreversible brain damage with serious consequences. Activation of oxidative stress and release of inflammatory mediators are considered potential pathological mechanisms. Butylphthalide (NBP) has anti-inflammatory and antioxidant effects on I/R injuries. However, it is unclear whether NBP can effectively mitigate renal I/R secondary to brain injury as well as its mechanism, which are the aims of this study. Both renal I/R injury rats and oxygen and glucose deprivation cell models were established and pre-intervened NBP. The Morris water maze assay was used to detect behavior. Hippocampal histopathology and function were examined after renal I/R. Apoptosis and tube-forming capacity of brain microvascular endothelial cells (BMVECs) were tested. Immunohistochemistry and Western blot were used to measure protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathway and NOD-like receptor C2 (NOD2)/Mitogen-activated protein kinases (MAPK)/Nuclear factor kappa-B (NF-κB) pathway. NBP treatment attenuated renal I/R-induced brain tissue damage and learning and memory dysfunction. NBP treatment inhibited apoptosis and promoted blood-brain barrier restoration and microangiogenesis. Also, it decreased oxidative stress levels and pro-inflammatory factor expression in renal I/R rats. Furthermore, NBP enhanced BMVECs’ viability and tube-forming capacity while inhibiting apoptosis and oxidative stress. Notably, the alleviating effects of NBP were attributed to Nrf2/HO-1 pathway activation and NOD2/MAPK/NF-κB inhibition. This study demonstrates that NBP maintains BBB function by activating the Nrf2/HO-1 pathway and inhibiting the NOD2/MAPK/NF-κB pathway to suppress inflammation and oxidative stress, thereby alleviating renal I/R-induced brain injury

Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

Renal ischemia-reperfusion (I/R) injury leads to irreversible brain damage with serious consequences. Activation of oxidative stress and release of inflammatory mediators are considered potential pathological mechanisms. Butylphthalide (NBP) has anti-inflammatory and antioxidant effects on I/R injuries. However, it is unclear whether NBP can effectively mitigate renal I/R secondary to brain injury as well as its mechanism, which are the aims of this study. Both renal I/R injury rats and oxygen and glucose deprivation cell models were established and pre-intervened NBP. The Morris water maze assay was used to detect behavior. Hippocampal histopathology and function were examined after renal I/R. Apoptosis and tube-forming capacity of brain microvascular endothelial cells (BMVECs) were tested. Immunohistochemistry and Western blot were used to measure protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathway and NOD-like receptor C2 (NOD2)/Mitogen-activated protein kinases (MAPK)/Nuclear factor kappa-B (NF-κB) pathway. NBP treatment attenuated renal I/R-induced brain tissue damage and learning and memory dysfunction. NBP treatment inhibited apoptosis and promoted blood-brain barrier restoration and microangiogenesis. Also, it decreased oxidative stress levels and pro-inflammatory factor expression in renal I/R rats. Furthermore, NBP enhanced BMVECs’ viability and tube-forming capacity while inhibiting apoptosis and oxidative stress. Notably, the alleviating effects of NBP were attributed to Nrf2/HO-1 pathway activation and NOD2/MAPK/NF-κB inhibition. This study demonstrates that NBP maintains BBB function by activating the Nrf2/HO-1 pathway and inhibiting the NOD2/MAPK/NF-κB pathway to suppress inflammation and oxidative stress, thereby alleviating renal I/R-induced brain injury.</p

Caspase-dependent apoptosis and necrosis inhibition.

<p>Z-VAD-FMK (20 µM and 100 µM) and Nec-1(50 µM, 100 µM and 200 µM) plus HMME (20 µg/mL) were added to cells 4 h before laser irradiation. <b>A.</b> Effects of caspase inhibitor on HMME-PDT mediated cell death. <b>B.</b> Effects of necrosis inhibitor on HMME-PDT mediated cell death. **P<0.05 was considered statistically significant by Student’s <i>t</i>-test and one-way analysis of variance. compared with the control group.</p

Activation of caspase-independent apoptotic pathway after HMME-PDT.

<p>The change of apoptosis related proteins was analyzed using western blotting. The relative expression levels of caspase-1, caspase-3, caspase-6, caspase-9 and PARP proteins were significantly up-regulated in HMME-PDT group. *P<0.05- was considered statistically significant by Student’s <i>t</i>-test, compared with the control group.</p

Effects of HMME-PDT on sarcoma cell viability by MTT assay.

<p>LM8, MG63, Saos-2, SW1353, TC71 and RD cells were cultured with different concentrations of HMME (10, 20 and 30 µg/mL) for 4 h and then irradiated by laser at different doses (3, 6, 9 J/cm²). The control group, PS group and laser group were selected as the control groups. The data were obtained from three independent experiments. *P<0.05 was considered statistically significant by student’s <i>t</i> -test, compared with the control group.</p

Immunohistochemistry (IHC) detection after HMME-PDT (×200).

<p>Tumors were cut and fixed with 10% formalin overnight and specimens were embedded in paraffin and cut into 4-µm-thick sections. Expression of caspase-1, caspase-3, caspase-6, caspase-9, and PARP proteins were evaluated by IHC.</p

Histological changes of tumor tissue post-PDT by H&E staining (×200).

<p>The necrotic areas of tumor dyes red isotropicly undergoing light microscopy. <b>A.</b> The tumor surface appeared the widespread necrosis and incrustation region in HMME-PDT group under gross appearance. <b>B.</b> The widespread necrotic areas and significant inflammatory cells infiltrative growth were observed in tumor tissues in the HMME-PDT group.</p

Intracellular uptake of HMME in C2C12, NIH/3T3, LM8, and K7 cells.

<p>The intracellular uptake of HMME was analyzed using flow cytometry with a PerCP-Cy5-5-A channel and evaluated based on fluorescence intensity. Fluorescence intensity on sarcoma cells (LM8 and K7)markedly increased with the incubation time and HMME concentrations. However, the fluorescence intensity of myoblast cells (C2C12) and fibroblast cells(NIH/3T3) was not significantly enhanced with increased incubation time or HMME concentration.</p