Tanreqing injection inhibits dengue virus encephalitis by suppressing the activation of NLRP3 inflammasome

Abstract

Background: Encephalitis caused by dengue virus (DENV) is considered a manifestation of severe dengue. Tanreqing injection (TRQ) is a well-known Chinese patented medicine, which has been used to treat brain-related disorders by inhibiting inflammation. Nevertheless, the effects of TRQ on DENV encephalitis have not been studied. The aim of this study was to evaluate the effects of TRQ on DENV encephalitis and to explore its potential mechanisms.

Methods: The cytotoxicity of TRQ was examined by MTT assay, and the anti-DENV activities of TRQ in BHK-21 baby hamster kidney fibroblast were evaluated through CCK-8 and plaque assays. The expression levels of NO, IL1B/IL-1β, TNFα and IL6 were measured by qRT‒PCR and ELISA in the BV2 murine microglial cell line. The inhibitory effects of TRQ on NLRP3 inflammasome activation in BV2 cells were examined by Western blotting, qRT‒PCR and ELISA. The effects of TRQ on HT22 mouse hippocampal neuronal cells were examined by CCK-8 assay, morphology observation and flow cytometry. Moreover, a DENV-infected ICR suckling mouse model was developed to investigate the protective role of TRQ in vivo.

Results: TRQ decreased the release of NO, IL6, TNFα and IL1B from BV2 cells and inhibited the activation of NLRP3. The presence of the NLRP3 agonist nigericin reversed the anti-inflammatory activities of TRQ. Furthermore, TRQ inhibited the death of HT22 cells by decreasing IL1B in DENV-infected BV2 cells. In addition, TRQ significantly attenuated weight loss, reduced clinical scores and extended the survival in DENV-infected ICR suckling mice. Critically, TRQ ameliorated pathological changes in ICR suckling mice brain by inhibiting microglia and NLRP3 activation and decreasing the production of inflammatory factors and the number of dead neurons.

Conclusion: TRQ exerts potent inhibitory effects on dengue encephalitis in vitro and in vivo by reducing DENV-2-induced microglial activation and subsequently decreasing the inflammatory response, thereby protecting neurons. These findings demonstrate the potential of TRQ in the treatment of dengue encephalitis.

Keywords: Dengue; Encephalitis; NLRP3; Tanreqing injection.

Fig. 1

TRQ exerts anti-DENV effects in vitro and in vivo. A The cytotoxicity of TRQ on BHK-21 cells. BHK-21 cells were incubated with TRQ for 96 h and cell viability was determined by MTT assay. B TRQ shows no prophylactic anti-DENV activities, as detected by CCK-8 assay. Cells were incubated with different concentrations of TRQ and then infected with DENV-2 for 1 h. C TRQ exerts anti-DENV attachment/entry effects on BHK-21 cells, as detected by CCK-8 assay. BHK-21 cells were infected with 0.5 MOI of DENV-2 with or without TRQ treatment. D TRQ shows no anti-DENV activities after entry into BHK-21 cells, as detected by CCK-8 assay. After infection with 0.5 MOI of DENV-2 for 1 h, BHK-21 cells were treated with TRQ. E TRQ inhibits the production of progeny viruses in DENV-2 infected BHK-21 cells as examined by plaque assay. F Statistical analysis of plaque assay to verify the inhibitory effects of TRQ on DENV- induced progeny virus. G, H TRQ inhibits the viral load in the brain in DENV-2 infected ICR suckling mice at 6 D.P.I., as determined by qRT‒PCR (n = 6). I–K TRQ downregulates the protein expression level of E protein in DENV-2 infected mouse brains, as examined by immunohistochemistry (100 ×) I, J and Western blotting K. The concentrations of TRQ-L, TRQ-M and TRQ-H were 1.25, 2.5 and 5 mL/kg, respectively. Dex was used as a positive drug. ^***P < 0.001 vs. control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. DENV-2 group

Fig. 2

TRQ attenuates the release of NO, IL6 and TNFα in DENV-2 infected murine microglial BV2 cells. A The cytotoxicity of TRQ in BV2 cells. BV2 cells were incubated with TRQ for 24 h and cell viability was determined by MTT assay. B–D TRQ inhibits the release of NO, IL6 and TNFα in BV2 cells. After exposure to DENV-2 (0.5 MOI) in the presence or absence of TRQ for 24 h, the secretion of NO (B), IL6 (C) and TNFα (D) was measured by NO assay kits or ELISA kits. ^*P < 0.05, ^***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. DENV-2 group

Fig. 3

TRQ decreases the activation of NLRP3 in DENV-2 infected BV2 cells. BV2 cells were infected with DENV-2 (0.5 MOI) in the presence or absence of TRQ (1/400, 1/200, 1/100) for 24 h. A, B TRQ downregulates the mRNA expression levels of Nlrp3 (A) and Il1b (B) in DENV-2 infected BV2 cells, as detected by qRT‒PCR. C–F TRQ decreases the protein expression levels of NLRP3 and cleaved caspase-1 in DENV-2 infected BV2 cells. Total proteins and supernatant proteins were extracted, and Western blotting was performed. G TRQ attenuates the release of IL1B in DENV-2 infected BV2 cells. The cell culture media were collected, and ELISA was performed to quantify the concentration of IL1B. H–J The activation of NLRP3 reverses the anti-inflammatory activities of TRQ in DENV-infected BV2 cells. BV2 cells were infected with DENV-2 (0.5 MOI) in the presence or absence of TRQ (1/100) for 22 h and then treated with 10 µM NLRP3 agonist nigericin for 2 h. The levels of IL1B (H) and cleaved CASP1 (I, J) in supernatants were analyzed by ELISA or Western blotting, respectively.^*P<0.05,  ^**P < 0.01, ^***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. DENV-2 group. ^$P < 0.05 vs. DENV-2 + TRQ group

Fig. 4

TRQ protects mouse hippocampal neuronal HT22 cells by decreasing IL1B from DENV-2-stimulated BV2 cells. A, B DENV-2 shows no obvious toxicities in HT22 cells. HT22 cells were incubated with DENV-2 for 24, 36 or 48 h and then examined by CCK-8 (A) and LDH (B) assays. C Steps of adoptive culture. BV2 cells were infected with DENV-2 (0.5 MOI or 5 MOI) in the presence or absence of TRQ for 24 h. Then, HT22 cells were incubated with cell-free supernatants from BV2 cells for 24 or 48 h. D, E TRQ protects HT22 cells from DENV-2-stimulated BV2 cells supernatants induced death. After adoptive culture, the morphological change and the cell viability of HT22 cells were examined by microscopy (40 ×) (D) and CCK-8 assay (E). F TRQ inhibits apoptosis in HT22 cells induced by supernatants from DENV-2-stimulated BV2 cells. After adoptive culture, the number of apoptotic HT22 cells was analyzed by PI/Annexin V-FITC staining. G–I IL1B counteracts the protective effect of TRQ. BV2 cells were infected with DENV-2 (0.5 MOI) in the presence or absence of TRQ for 24 h. Then, HT22 cells were incubated with cell-free supernatants from BV2 cells in the presence or absence of IL1B for 48 h. Morphological observation of cell viability and apoptosis of the HT22 cells were performed by microscopy (40 ×) (G), CCK-8 assay (H) and PI/Annexin V-FITC staining (I). ^***P < 0.001 vs. control group, ^##P < 0.01, ^###P < 0.001 vs. DENV-2 group, ^$$$P < 0.001 vs. DENV-2 + TRQ group, ⁺⁺⁺P < 0.001 vs. DENV-2 + TRQ + IL1B group

Fig. 5

TRQ inhibits encephalitis in DENV-infected ICR suckling mice. Seven-day-old ICR suckling mice were infected with DENV-2 by intracranial injection, and TRQ (1.25, 2.5, 5.0 mL/kg) was administered by intraperitoneal injection. The body weights (A), disease manifestations (B) and survival rates (C) of mice were monitored daily (n = 6). Disease manifestations were scored as follows: 0 for health, 1 for minor symptoms (reduced mobility and hunched posture), 2 for limbic seizure, 3 for moving difficulties (anterior or posterior limb weakness), 4 for paralysis and 5 for death. D–F TRQ inhibits the release of NO, IL6 and TNFα. After mice were sacrificed, the levels of NO, IL6 and TNFα in the brain were measured by NO or ELISA kits (n = 6). G TRQ ameliorates the brain tissue lesions caused by DENV-2. The brain tissues were dehydrated and embedded in paraffin. After sectioning a thickness of 4 μm, brain pathological changes were detected by HE staining (100 ×). H TRQ decreases neuronal injury caused by DENV-2 detected by Nissl staining (100 ×). I TRQ inhibits the microglial activation in DENV-infected ICR suckling mice, as examined by immunohistochemistry. Representative image of Iba-1 immunostaining of Iba-1 in the brain (100 ×). J TRQ reduces DENV-2-induced apoptosis in neurons. Apoptosis was detected by Hoechst 33258 staining (100 ×). ^*P < 0.05, ^***P < 0.001 vs. control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. DENV group

Fig. 6

TRQ inhibits the activation of NLRP3 in brain tissues of DENV-infected mice. A, B TRQ decreases the mRNA expression levels of Nlrp3 (A) and Il1b (B) in DENV-infected mice (n = 6). The brain tissues of the mice were harvested and total mRNAs were extracted and analyzed by qRT‒PCR. C–E TRQ decreases the protein expression level of CASP1 in DENV-infected mice. The brain tissues of the mice were harvested and total protein lysates were collected and analyzed by Western blotting. F TRQ decreases the release of IL1B in DENV-infected mice. The level of IL1B in the brain was measured by ELISA (n = 6). G TRQ inhibits NLRP3 in DENV-infected mice. The brain tissues were sectioned and the expression of NLRP3 in the mouse brain tissues was determined with anti-NLRP3 antibodies by immunofluorescence staining (200 ×). DAPI was used to stain the nucleus. ^**P < 0.01, ^***P < 0.001 vs. control group, ^#P < 0.05, ^##P < 0.01 vs. DENV group. (F) The mechanism of TRQ on dengue encephalitis. The schematic was drawn with BioRender (https://biorender.com/)