Lupeol alleviates coxsackievirus B3-induced viral myocarditis in mice via downregulating toll-like receptor 4

Figures

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  Figure 1.. Effects of lupeol on myocardial histopathology and survival rates. (a) Cardiac injury as demonstrated by haematoxylin and eosin (H&E) staining (magnification 400×). (b) Survival rate of the four groups of mice. (c) Myocarditis scores (mean ± SD) of the four groups of mice. Foci of mononuclear infiltration and myocardial necrosis were quantified from 5 high-power fields per section and each specimen was graded as follows: 0: no inflammation or injury; 1: <5% involvement; 2: 5% – 25% involvement; 3: 25% – 75% involvement; 4: >75% involvement. DMEM (Dulbecco's Modified Eagle Medium) treated mice (n = 20); CVB3 (coxsackie virus B3) infected mice (n = 40); EXP-L mice (CVB3 + 50 mg/kg lupeol [n = 40]); EXP-H mice (CVB3 + 100 mg/kg lupeol [n = 40]). **P < 0.01 compared with DMEM group, ##P < 0.01 compared with CVB3 group.

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  Figure 2.. Effect of lupeol on virus titres and myocardial damage markers. (a) Virus titres; (b) cardiac troponin I (cTn I) levels; (c) muscle haemoglobin (Mb) levels; (d) MB isoenzyme of creatine kinase (CK-MB) levels. Values are shown as mean ± SD. DMEM treated mice (n = 20); CVB3 (coxsackie virus B3) infected mice (n = 40); EXP-L mice (CVB3 + 50 mg/kg lupeol [n = 40]); EXP-H mice (CVB3 + 100 mg/kg lupeol [n = 40]). **P < 0.01 compared with DMEM group, ##P < 0.01 compared with CVB3 group. TCID, tissue culture infective dose.

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  Figure 3.. Effect of lupeol on pro-inflammation cytokines. (a) Levels of interleukin (IL)-β and tumour necrosis factor alpha (TNF-α) were detected by commercially available enzyme-linked immunosorbent (ELISA) kits. Values are shown as mean ± SD. (b) Expression of cyclooxygenase-2 (COX2) was determined by reverse transcription polymerase chain reaction (RT-PCR). Values are shown as mean ± SD. (c) Expression of interleukin (IL)-6 detected by immunohistochemical staining. (d) Percentage of IL-6 positive cells shown by immunohistochemical staining. Values are shown as mean ± SD. DMEM treated mice (n = 20); CVB3 (coxsackie virus B3) infected mice (n = 40); EXP-L mice (CVB3 + 50 mg/kg lupeol [n = 40]); EXP-H mice (CVB3 + 100 mg/kg lupeol [n = 40]); CVB3-siRNA mice (CVB3 + small interfering RNA (siRNA)- toll-like receptor 4 (TLR4), [n = 20]); siRNA + EXP-H mice (CVB3 + siRNA-TLR4 + 100 mg/kg lupeol [n = 20]); **P < 0.01 compared with DMEM group, ##P < 0.01 compared with CVB3 group.

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  Figure 4.. Effect of lupeol on the expression of toll-like receptor (TLR) 4, MyD88, phosphorylated NF-κB P65 [P-NF-κb-P65] and NF-κB P65. (a) The protein expressions of TLR4, MyD88, NF-κB P65and p-NF-κB P65 determined by western blotting. (b) mRNA levels of TLR4 determined by the reverse transcription polymerase chain reaction (RT-PCR). Values are shown as mean ± SD. (c–e) Quantitative analysis of the expression of TLR4, MyD88, NF-κB P65and p-NF-κB P65. Values are shown as mean ± SD. DMEM treated mice (n = 20); CVB3 (coxsackie virus B3) infected mice (n = 40); EXP-L mice (CVB3 + 50 mg/kg lupeol [n = 40]); EXP-H mice (CVB3 + 100 mg/kg lupeol [n = 40]). **P < 0.01 compared with DMEM group, ##P < 0.01 compared with CVB3 group.