DHLA treatment reversed the LPS-induced sickness behaviors
Body weight gain and behavioral tests, including OFT and FST were performed to investigate the effects of DHLA on LPS-induced sickness behaviors in rats.
As shown in Fig. 2A, the Shapiro-Wilk test results showed that the data are normally distributed (p > 0.05). Rats exposed to LPS showed less body weight gain as compared to the control group (F(5,30) = 19.83, p < 0.0001). However, treatment with DHLA (30 mg/kg, p < 0.001; 60 mg/kg, p < 0.01) and Flu (p < 0.0001) improved the body weight gain as compared to the LPS group.
The FST is mainly used to measure the depression-like behavior. As shown in Fig. 2B, the Shapiro-Wilk test results showed that the data are normally distributed (p > 0.05). Rats exposed to LPS showed more immobility time in FST as compared to the control group (F(5,30) = 17.49, p < 0.0001), whereas compared to the LPS group, DHLA (30 mg/kg, p < 0.001; 60 mg/kg, p < 0.01) and Flu (p < 0.0001) treatment markedly decreased the immobility time in FST.
The performance of rats in OFT is shown in Fig. 2C–E. The Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The total distance, total velocity, and rearing frequencies were significantly decreased in the LPS group as compared to the control group (total distance (F(5,30) = 19.93, p < 0.0001); velocity (F(5,30) = 8.132, p < 0.0001); rearing frequencies (F(5,30) = 11.66, p < 0.0001). Compared to the LPS group, the total distance in the DHLA (30 mg/kg, p < 0.001; 60 mg/kg, p < 0.01) and Flu (p < 0.0001) groups was significantly increased, the total velocity in the DHLA (30 mg/kg, p < 0.01; 60 mg/kg, p < 0.05) and Flu (p < 0.01) groups were significantly increased, and the rearing frequencies in the DHLA (30mg/kg, p < 0.01) and Flu (p < 0.01) groups were significantly increased.
Since DHLA (30 mg/kg) was superior to the other doses in measurements (Fig. 2A-E), we selected 30 mg/kg as the optimal dose of DHLA and used it in the following experiments.
DHLA inhibited the activation of microglia induced by LPS
To measure the number of microglia cells, immunofluorescence staining was performed by using the microglia-specific marker, IBA-1. As shown in supplementary materials Fig. S3 A-B, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). Rats exposed to LPS showed more number microglia as compared to the control group (F(4,25) = 22.16, p < 0.001). However, treatment with DHLA (p < 0.01) decreased the microglia cells number as compared to the LPS group.
DHLA reversed the LPS-induced sickness behaviors through ERK/Nrf2/HO-1/ROS/NLRP3-dependent inflammation pathway
Western blot and immunofluorescence staining were used to test the expression of ERK/Nrf2/HO-1/ROS/NLRP3 signaling pathway in response to DHLA against LPS-induced sickness behaviors in rats.
As shown in Fig. 3A–D, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). A statistically significant difference between groups for p-ERK (F(3,20) = 13.97, p < 0.0001), Nrf2 (F(3,20) = 9.48, p < 0.0001), and HO-1 (F(3,20) = 10.90, p < 0.0001) was determined by one-way ANOVA. Tukey’s post-hoc analysis revealed that expression levels of p-ERK (p < 0.01), Nrf2 (p < 0.01), and HO-1 (p < 0.01) were significantly higher in DHLA-treated rats as compared to the LPS groups. Immunofluorescence staining revealed that the expression of HO-1 in the DHLA group was increased than that in the control group (Fig. 3E). As shown in Fig. 4A–D, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). A statistically significant difference in NLRP3 (F(3,20) = 21.62, p < 0.0001), caspase-1 (F(3,20) = 17.37, p < 0.0001), and IL-1β(F(3,20) = 11.93, p < 0.0001) between the groups as determined by one-way ANOVA. Tukey’s post-hoc analysis revealed that the expression levels of NLRP3 (p < 0.0001), caspase-1 (p < 0.0001), and IL-1β (p < 0.001) were significantly higher in LPS rats as compared to the control groups. However, these effects were significantly reversed by the treatment with DHLA (NLRP3, p < 0.01; caspase-1, p < 0.01; IL-1β, p < 0.05).
To verify the effect of DHLA alone on ERK/Nrf2/HO-1/NLRP3 signaling pathway, we designed another four groups. As shown in supplementary materials Fig. S2, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). A statistically significant difference between groups for p-ERK (F(3,20) = 30.09, p < 0.0001), Nrf2 (F(3,20) = 10.57, p < 0.0001), HO-1 (F(3,20) = 12.03, p < 0.0001) , NLRP3 (F(3,20) = 28.55, p < 0.0001), and IL-1β (F(3,20) = 16.58, p < 0.0001) was determined by one-way ANOVA. Tukey’s post-hoc analysis revealed that expression levels of p-ERK (p < 0.01), Nrf2 (p < 0.01), and HO-1 (p < 0.01) were significantly higher in DHLA alone group as compared to the LPS groups, and the expression levels of NLRP3 (p < 0.01), and IL-1β (p < 0.05) were significantly lower in DHLA alone group as compared to the LPS groups.
The concentration of intracellular ROS was evaluated by the changes in DCF fluorescence intensity using flow cytometry. As shown in Fig. 4E, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The ROS expression was distinctly higher in the LPS rats as compared to the control group (F(3,20) = 25.80, p < 0.0001). Conversely, the intracellular ROS level induced by LPS was markedly reversed by DHLA (p < 0.001).
Body weight gain and behavioral tests, including OFT and FST, were performed to further investigate the effects of DHLA on LPS-induced sickness behaviors in rats. As shown in supplementary materials Fig. S1 A-E. The Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). As shown in supplementary materials Fig. S1A, rats exposed to LPS showed less body weight gain than the control group (F(5,30) = 20.84, p < 0.0001). However, treatment with DHLA (30 mg/kg, p < 0.01) improved the body weight gain as compared to the LPS group. Fig. S1B showed that rats exposed to LPS showed more immobility time in FST as compared to the control group (F(5,30) = 27.11, p < 0.0001). On the other hand, compared to the LPS group, DHLA (30 mg/kg, p < 0.001) treatment markedly decreased the immobility time in FST. As shown in Fig. S1C–E, the total distance, total velocity, and rearing frequencies were significantly decreased in the LPS group as compared to the control group (total distance (F(5,30) = 30.48, p < 0.0001); velocity (F(5,30) = 12.16, p < 0.0001); rearing frequencies (F(5,30) = 15.22, p < 0.0001). Compared to the LPS group, the total distance, total velocity, and rearing frequencies in the DHLA (30 mg/kg, all p < 0.01) group were significantly increased.
Blockade of ERK abolished the preventive effect and anti-inflammation effect of DHLA
Previous studies have suggested that the ERK pathway is related to the regulation of Nrf2 [29]. PD98059 (an ERK pathway inhibitor) was administered in LPS-induced sickness behaviors rats 1 h before the administration of DHLA to investigate whether DHLA upregulated the expression of Nrf2/HO-1 in LPS-induced rats via the ERK pathway.
As shown in Fig. 5A, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). Rats exposed to DHLA showed higher body weight gain as compared to the LPS group (F(5,30) = 22.61, p < 0.0001). However, treatment with PD98059 (p < 0.01) decreased body weight gain than the DHLA group.
As shown in Fig. 5B–E, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The DHLA administration greatly attenuated the depression-like behavior in LPS-induced rats. As shown in Fig. 5B, rats exposed to DHLA showed less immobility time in FST as compared to the LPS group (F(5,30) = 26.79, p < 0.0001), whereas compared to the DHLA group, the PD98059 (p < 0.05) treatment markedly increased the immobility time in FST. As shown in Fig. 5C–E, the total distance, rearing frequencies, and total velocity were significantly increased in the DHLA group as compared to the LPS group (total distance F(5,30) = 22.78, p < 0.0001; rearing frequencies F(5,30) = 18.71, p < 0.0001; velocity F(5,30) = 15.17, p < 0.0001). However, compared to the DHLA group, the total distance in the PD98059 (p < 0.01) group and rearing frequencies and total velocity in the PD98059 (both p < 0.05) group were significantly decreased. These data indicated that the preventive effect of DHLA in the LPS-induced rats was blocked by PD98059.
As shown in Fig. 6A–F, the Shapiro-Wilk test results showed that the western blot data are all normally distributed (p > 0.05). The results showed a statistically significant difference between the study groups as determined by one-way ANOVA with respect to Nrf2 (F(5,30) = 35.07, p < 0.0001), HO-1 (F(5,30) = 25.19, p < 0.0001), NLRP3(F(5,30) = 50.79, p < 0.0001), caspase-1 (F(5,30) = 15.64, p < 0.0001), and IL-1β (F(5,30) = 34.71, p < 0.0001). Tukey’s post-hoc analysis revealed that expression levels of Nrf2 (p < 0.0001) and HO-1 (p < 0.0001) were significantly higher in DHLA rats as compared to the LPS groups. In addition, the expression of NLRP3 (p < 0.0001), caspase-1 (p < 0.01), and IL-1β (p < 0.0001) was significantly lower in DHLA rats as compared to the LPS groups. However, inhibition of ERK with PD98059 abolished the effects of DHLA, which led to decreased Nrf2 (p < 0.01) and HO-1 (p < 0.01), while increased in the inflammation-related proteins, such as NLRP3 (p < 0.01), caspase-1 (p < 0.05), and IL-1β (p < 0.01) as compared to the DHLA group.
As shown in Fig. 6G, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The ROS expression was distinctly lower in the DHLA group as compared to the LPS group (F(5,30) = 23.09, p < 0.0001). Conversely, the ROS level induced by DHLA was markedly ameliorated by PD98059 (p < 0.01).
Blockade of Nrf2 abolished the preventive effect and anti-inflammation effect of DHLA
Body weight gain and behavioral tests showed that AAV-Nrf2-siRNA completely abolished the treatment effects of DHLA in the LPS-induced sickness behaviors rats.
As shown in Fig. 7A, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). Rats exposed to DHLA showed higher body weight gain as compared to the LPS group (F(5,30) = 17.46, p < 0.0001). However, treatment with AAV-Nrf2-siRNA (p < 0.05) decreased body weight gain as compared to the DHLA group.
As shown in Fig. 7B–E, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The DHLA administration greatly attenuated the sickness behaviors observed in LPS-induced rats. As shown in Fig. 7B, rats exposed to DHLA showed less immobility time in FST as compared to the LPS group (F(5,30) = 24.1, p < 0.0001). On the other hand, compared to the DHLA group, the AAV-Nrf2-siRNA (p < 0.05) treatment markedly increased the immobility time in FST. As shown in Fig. 7C–E, the total distance, rearing frequencies, and total velocity were significantly increased in the DHLA group that in the LPS group (total distance F(5,30) = 17.84, p < 0.0001; rearing frequencies F(5,30) = 17.87, p < 0.0001; velocity F(5,30) = 13.86, p < 0.0001). However, compared to the DHLA group, the total distance, the rearing frequencies and total velocity in the DHLA (p < 0.05, p < 0.01, p < 0.05, respectively) group were significantly decreased. These data indicated that the preventive effect of DHLA in LPS-induced rats was blocked by AAV-Nrf2-siRNA.
As shown in Fig. 8A–E, the Shapiro-Wilk test results showed that the western blot data are all normally distributed (p > 0.05). The results showed a statistically significant difference between the study groups as determined by one-way ANOVA regarding Nrf2 (F(5,30) = 11.77, p < 0.0001), HO-1 (F(5,30) = 41.88, p < 0.0001), NLRP3 (F(5,30) = 23.01, p < 0.0001), caspase-1 (F(5,30) = 20.75, p < 0.0001), and IL-1β (F(5,30) = 25.14, p < 0.0001). Tukey’s post-hoc analysis revealed that the expression levels of Nrf2 (p < 0.01) and HO-1 (p < 0.0001) were significantly higher in DHLA rats as compared to that in the LPS groups. Moreover, the expression of NLRP3 (p < 0.001), caspase-1 (p < 0.001), and IL-1β (p < 0.0001) was significantly lower in DHLA rats as compared to the LPS groups. However, inhibition of Nrf2 with AAV-Nrf2-siRNA abolished the effects of DHLA, which led to a decrease in Nrf2 (p < 0.05) and HO-1 (p < 0.01) expression and increase in the expression of inflammation-related proteins NLRP3 (p < 0.01), caspase-1 (p < 0.01), and IL-1β (p < 0.001) as compared to the DHLA group.
As shown in Fig. 8G, the Shapiro-Wilk test results showed that the data are all normally distributed (p > 0.05). The results showed that the ROS expression was distinctly lower in the DHLA group as compared to the LPS group (F(5,30) = 14.89, p < 0.0001). In contrast, the ROS level induced by DHLA was markedly ameliorated by AAV-Nrf2-siRNA (p < 0.05).