| Trichinella spiralis(Ts)is a widely distributed zoonotic parasite worldwide.When severely infected with Trichinella spiralis,it can cause symptoms such as systemic muscle soreness,fever,nausea,diarrhea,vomiting,and papules in the host.It is a type of parasite that endangers the safety of animal husbandry production and human health,especially in low-income countries and regions with high infection rates.The growth and development of Trichinella spiralis require continuous shuttling through the small intestinal villi,which can cause serious damage to it.Therefore,the host organism will initiate corresponding worm expulsion reactions to accelerate the expulsion of the parasite.The organism also has a similar reaction response to the invasion of other intestinal parasitic worms,such as accelerating the contraction frequency of the small intestine.These reactions are all part of the organism’s response to worm explosion invasion of intestinal parasitic worms,which can reduce the damage of intestinal parasites to intestinal villi and accelerate the elimination of intestinal parasites.5-HT is a neurotransmitter widely distributed in the intestines of humans and animals.It can regulate the contraction of intestinal smooth muscle,thereby affecting the movement and physiological state of the intestines,to increase intestinal peristalsis and promote the elimination of intestinal parasite.Research has shown that intestinal parasitic infections can also alter the secretion level of 5-HT in the intestine.IL-33 is one of the warning factors in the intestine,which is released by epithelial cells upon receiving external stimuli.IL-33 in the intestine can be received by transmembrane ST2 receptor to form an IL-33-ST2 signal,which is found in both hematopoietic and non hematopoietic cells.This signal is a key signal that controls the release of 5-HT by enterochromaffin cells(EC).Therefore,this study aims to explore the secretion regulation mechanism of 5-HT during the intestinal phase of Trichinella spiralis infection.By establishing a model of Trichinella spiralis infection and measuring the changes in the secretion level of 5-HT in the intestinal phase,combined with relevant physiological indicators and histological detection results,we explore the secretion regulation mechanism of5-HT in the intestinal phase of Trichinella spiralis infection.This article is hoped to provide new ideas for revealing the mechanism of worm expulsion reaction in the intestinal phase of Trichinella spiralis infection.The research content and results are as follows:(1)Secretion and expression status of 5-HT and IL-33-ST2 signaling pathway related proteins during the intestinal phase of Trichinella spiralis infectionA total of 24 C57BL/6 mice were divided into an insect attack group of 18 and a control group of 6.Each mouse in the attack group was given 300 Trichinella spiralis muscle larvae by gavage,and small intestine samples,mesenteric lymph node samples,spleen samples,serum samples,and fecal diluent supernatant samples were collected on the 3rd,7th,and 17 th days after collecting the insect attack group.Subsequently,tissue samples were subjected to Q-PCR and Western blot detection,and fecal diluent supernatant,as well as serum samples were conducted ELISA detection.The Q-PCR results showed that the transcription level of IL-33 in the intestinal tissue of mice infected with Trichinella spiralis significantly increased on the 3rd day after infection(p<0.001).The ST2 transcription level significantly increased on the 17 th day after infection(p<0.001),and the TPH1 transcription level significantly increased on the 17 th day after infection(p<0.01).The Western blot results were similar to the Q-PCR results.The expression level of IL-33 protein significantly increased on the 3rd day after Trichinella spiralis infection(p<0.001).The expression level of ST2 protein significantly increased on the 3rd day after Trichinella spiralis infection(p<0.01).The ELISA results showed that the 5-HT secretion in the fecal diluent supernatant significantly increased on the 7th day after Trichinella infection,and IL-33 significantly increased on the 3rd day after Trichinella infection(p<0.01),but there was no significant difference in serum(p>0.05).(2)The effect of inhibiting ST2 protein on 5-HT secretion levelIn this experiment,the cells were divided into three groups: NC group,i ST2+TsAES group,and Ts AES group.The NC group was not treated,and the i ST2+Ts-AES group cells were inhibited by i ST2 for 24 hours.The i ST2+Ts-AES group and Ts-AES group cells were then stimulated with Ts-AES for 24 hours.Collect supernatant from each group of cell culture medium for ELISA detection,and collect cells from each group for Q-PCR detection.Q-PCR detection results showed that the ST2 transcription level in the Ts-AES group was significantly increased compared to the NC group(p<0.05),the ST2 transcription level in the i ST2+Ts-AES group was significantly reduced compared to the NC group(p<0.05),and the IL-33 transcription level in the Ts-AES group,as well as i ST2+Ts-AES group was significantly increased compared to the NC group(p<0.01),The TPH1 transcription level in the i ST2+TsAES group was significantly reduced compared to the NC group(p<0.05),but there was no significant change in the TPH1 transcription level in the Ts-AES group(p>0.05).The ELISA results showed that in the supernatant of cell culture medium,the secretion volume of IL-33 in the i ST2+Ts-AES group was significantly increased compared to the NC group(p<0.01),while the secretion volume of IL-33 in the TsAES group was significantly increased compared to the NC group(p<0.05).The results of 5-HT content were used for immunofluorescence detection of cells in each group.The Q-PCR detection results showed an increase in IL-33 secretion by MODEK cells and a decrease in 5-HT transcription levels.The results of ELISA detection of cell culture medium supernatant showed a significant decrease in 5-HT secretion levels in the i ST2+Ts-AES group compared to the NC group(p<0.01).Afterwards,24 C57BL/6 mice were divided into a control group of 6 and an inhibitor insect attack group of 18.The inhibitor insect attack group was intervened with inhibitors,and i ST2 was started to inject intraperitoneally 6 days before the Trichinella spiralis attack,with 100μL injected intraperitoneally every two days.On the 6th day after the inhibitor injection intervention,300 Trichinella spiralis muscle larvae were gavaged to the mice in the inhibitor insect attack group.The samples of small intestine,spleen,and mesenteric lymph nodes from mice infected with Trichinella spiralis were collected on the 3rd,7th,and 17 th days after infection in the inhibitor insect attack group.The control group also collected the above samples for detection,analysis of IL-33-ST2 signaling pathway related gene transcription levels and protein secretion expression using Q-PCR,ELISA,as well as immunofluorescence methods.Adult worm counts were also performed on the 3rd day of Trichinella spiralis infection.The Q-PCR results showed that the IL-33 gene transcription level in mice significantly increased on the 3rd day after infection with Trichinella spiralis under the influence of the inhibitor(p<0.01),while the ST2 gene transcription level only significantly increased on the 7th day after infection(p<0.05)The TPH1 gene transcription level did not show significant differences in the intestinal phase of Trichinella spiralis(p>0.05).The ELISA results showed that there was no significant difference in the secretion volume of 5-HT during the intestinal phase of Trichinella spiralis infection under the action of i ST2(p>0.05).On the 7th day after Trichinella spiralis infection,the secretion volume of IL-33 significantly increased in the fecal diluent supernatant(p<0.05).The immunofluorescence results showed that under the influence of i ST2,the expression of ST2 protein in the intestinal phase of Trichinella spiralis infection increased significantly only on the 3rd day after infection(p<0.05),and the secretion and expression status of IL-33 increased significantly on the 3rd day after infection(p<0.01).However,ST2 decreased significantly on the 7th and 17 th days after infection(p<0.05).The adult worm count results showed that under the intervention of the inhibitor,the adult worm count of the inhibitor insect attack group increased by 59.64% compared to the number of the Trichinella spiralis group.This article proves that the intestinal infection stage of Trichinella spiralis stimulates the expression of IL-33-ST2 signals,thereby promoting the secretion of 5-HT in the intestine.5-HT stimulates the contraction of intestinal smooth muscle,to promote worm expulsion.This research provides a new approach for the study of the worm expulsion reaction of Trichinella spiralis infection. |
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