Effect of oxymatrine on Toll-like receptors on intestinal mucosa of mice infected by Cryptosporidium parvum

Abstract

Abstract:

Objective To study the effects of oxymatrine (OMT) on Toll-like receptors 2 (TLR2) and Toll-like receptors 4(TLR4) on intestinal mucosa of mice infected with Cryptosporidium parvum and investigate the molecular mechanisms of cryptosporidiosis treatment by OMT. Methods Thirty healthy BALB/c male mice were randomly divided into the infection group, OMT treatment group and non-infection group(n = 10 in each group). Mice in the infection group and OMT treatment group received intragastrical inoculation of C. parvum oocysts (1 × 10⁵/gram) to establish the model of C. parvum intestinal infection, while mice in the non-infection group were fed with normal diet and water. Mice in the OMT treatment group received daily intragastrical inoculation of 50 mg/kg for 2 weeks. The numbers of mean oocysts per gram of mouse feces were counted using the auramine-phenol modified anti-acid staining method. All mice were sacrificed after 2 weeks of treatment. The pathological changes of intestinal mucosa were observed by HE staining, and the height of intestinal villi and the depth of crypt were measured. RNA was extracted from intestinal mucosa, and transcribed into cDNA. The relative expression of TLR2 mRNA and TLR4 mRNA in intestinal mucosa was assessed by real-time fluorescence PCR. The relative expression of TLR2 and TLR4 in intestinal mucosa was assessed by Western blotting. Result The auramine-phenol modified anti-acid staining revealed increased numbers of mean oocysts in mouse feces in the infection group, peaking at day 7 with（179.24 ± 21.12） oocysts/gram, infection intensity was level 4, and reaching a plateau afterwards. In the OMT treatment group, the numbers decreased from day 5, and infection intensity was approaching level 0 after 2 weeks of treatment. HE staining showed atrophy and shedding of intestinal villi in the infection group, with submucosal edema and a significant gap between muscle layers, while the intestinal villi were intact in the non-infection group. The intestinal villi were restored in the OMT treatment group. The villi heights in the non-infection group, infection group and OMT treatment group were (348.1 ± 18.2), (278.0 ± 52.9) and (346.1 ± 19.2) μm, respectively, OMT treatment group was higher than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). The crypt depths in the non-infection group, infection group and OMT treatment group were (149.9 ± 27.4), (173.1 ± 11.1) and (155.4 ± 5.3) μm, OMT treatment group was lower than infection group (P < 0.05), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). The ratios of villi height/crypt depth in the non-infection group, infection group and OMT treatment group were 2.4 ± 0.6, 1.6 ± 0.3 and 2.2 ± 0.2, OMT treatment group was higher than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). qRT-PCR showed that the relative expression of TLR2 mRNA in intestinal mucosa were 1.0 ± 0.0, 3.0 ± 0.1 and 1.4 ± 0.3 in the non-infection group, infection group and OMT treatment group, and that of TLR4 mRNA were 1.0 ± 0.0, 3.1 ± 0.3 and 1.5 ± 0.1, respectively. That of OMT treatment group was lower than infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). Western blotting showed that the relative expression of TLR2 in the intestinal mucosa were 0.2 ± 0.1, 0.6 ± 0.1 and 0.2 ± 0.0, and that of TLR4 were 0.2 ± 0.1, 0.6 ± 0.0 and 0.3 ± 0.1 in the non-infection group, infection group and OMT treatment group respectively. That of OMT treatment group was lower than that of infection group (P < 0.01), and there was no significant difference between OMT treatment group and non-infection group (P > 0.05). Conclusion OMT improves intestinal mucosal repair through down-regulating TLR2 and TLR4 expression in mice infected with C. parvum.

Key words: Cryptosiporidium parvum, Toll-like receptors, Oxymatrine

CLC Number:

R382.3

Rui JI, Rui-wen LIANG, Zhi-yu GUAN, Rui-fang LI, Yu-rong FU, Hong-yan WANG. Effect of oxymatrine on Toll-like receptors on intestinal mucosa of mice infected by Cryptosporidium parvum[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(5): 455-460.

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引物Primer	序列(5′→3′) Sequence (5′→3′)
TLR2	F：GTGCCCTGTGCCACCATT
TLR2	R：GGAACGAAGTCCCGCTTGT
TLR4	F：GGGCTCATTCACTCACTAACGG
TLR4	R：CACAGGAGGGACCATCTTCATT
GAPDH	F：GAGCCAAAAGGGTCATCATCT
GAPDH	R：AGGGGCCATCCACAGTCTTC