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1、Schistosome infection improves hepatic insulin sensitivity through downregulating miR-802 expression Zhang fan1, Luo xiaofeng1, Zhang wenyue1, Yang bingya1,2, Hou min1, Liu ran1, Chen lin1,2, Ji Minjun1,2 1. Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, China 2 Jiangs
2、u Province Key Laboratory of Modern Pathogen Biology, Nanjing, Jiangsu, China * Corresponding authors: Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, 210029, P.R. China. Tel (Fax): +86-25-86862793. E-mail: Abstract Schistosomiasis japonica is a beast of parasitic dise
3、ases. The main pathological mechanism is the local chronic inflammation in liver and intestine triggered by eggs and tissue damage caused by over-repair of chronic infection, and development into liver fibrosis. Schistosomes are regarded as detrimental to humans, however, some studies have shown tha
4、t schistosomes not only cause damage to the body, but also can reduce the rate of some autoimmune diseases and metabolic diseases. Chronic inflammation associated with obesity contributes to some metabolic syndromes, such as insulin resistance and type 2 diabetes. The type 2 immune responses induced
5、 by Schistosoma mansoni infection and egg antigens immunization were reported to improve the whole-body glucose tolerance and insulin sensitivity, and tissue-specific insulin sensitivity of adipose tissue in obese mice. As Schistosoma japonicum and Schistosoma mansoni have many similarities in biolo
6、gical characters, pathogenesis and pathophysiology changes etc. We conceived that Schistosoma japonicum infection could improve whole-body and tissue-specific insulin sensitivity in type 2 diabetes. We established Schistosoma japonicum (S. japonicum) infection mice model with BALB/c, C57BL/6 and Lep
7、rdb/db male mice in order to explore the relationship between schistosome infection and energy metabolism. Six experimental groups were designed as the BALB/c control group (BALB/c-con), BALB/c infected group (BALB/c-inf), C57BL/6 control group (C57BL/6-con), C57BL/6 infected group (C57BL/6-inf), Le
8、prdb/db control group (Leprdb/db-con) and Leprdb/db infected group (Leprdb/db-inf). Glucose tolerance test and insulin tolerance test were used to evaluate body insulin sensitivities. Next, we used automatic biochemical analyzer to test serum alanine aminotransferase (ALT), aspartate aminotransferas
9、e (AST), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). HE staining to evaluate the degree of liver inflammation, oil red staining to determine the fat deposition, enzyme-linked immunosorbent assay (ELISA) to
10、measure serum insulin concentration, real-time fluorescent quantitative PCR to detect the transcription level of miR-802, Hnf1b and some inflammation related genes. The main results of our studies were as follows: 1. miRNA transcriptome in murine liver with S. japonicum infection showed that compare
11、d with normal mice, some miRNAs were differentially expressed in S. japonicum infected group, such as miR-155, miR-146b and miR-802. 2. Liver miR-802 expression was reduced and its target gene Hnf1b mRNA transcription was elevated after schistosome infection. Thus, S. japonicum infection might suppr
12、ess the expression of miR-802 in host liver and weaken its inhibitory function of Hnf1b. 3. The fasting glucose in S. japonicum infected mice were decreased compared with the controls. After glucose loading, blood glucose concentrations in S. japonicum infected C57BL/6 and Leprdb/db mice were persis
13、tently lower than those in their respective controls, especially in Leprdb/db mice. These suggested that S. japonicum infection induced glucose intolerance in C57BL/6 and Leprdb/db mice. 4. The results of HE staining and oil red staining in the liver showed that there were little fat in the liver of
14、 C57BL/6 mice. Compared with Leprdb/db control group, the density and area of fat in the liver of Leprdb/db infected group were significantly decreased. 5. ELISA results showed that the serum insulin concentration of infected mice were decreased compared with the respective controls. 6. Compared wit
15、h the respective control group, the levels of serum TC, TG, HDL-L, LDL-L in mice of S. japonicum infected group dropped significantly . 7. RT-PCR results showed that hepatic miR-802 expression in infected group was significantly decreased and mRNA level of Hnf1b was increased. More significant chang
16、e was observed in Leprdb/db mice. 8. RT-PCR results showed that mRNA levels of some proinflammatory and anti-inflammatory cytokines, including TNF-, IL-6, IL-22 and IL-4, in 6w infected group were significantly higher than those in the control group. Leprdb/db mice showed more obvious change. 9. In
17、vitro results showed that miR-802 expression in murine hepatocytes line were downregulated with IL-22 stimulation and upregulated with TNF- treatment. To sum up, we found that Schistosoma japonicum infection could improve hepatic insulin sensitivity possibly through downregulating miR-802 expression
18、 in obese mice. These experimental evidences may provide further guide in the prevention and control of type 2 diabetes mellitus. Background It is well recognized by many scientists that helminths infection can alleviate some autoimmune diseases (type 1 diabetes, multiple sclerosis etc) based on the
19、 epidemiological data (we call it hygiene hypothesis)1, 2 and some laboratory results3-5. One important reason might be that the regulatory factors derived from helminths tend to induce type 2 immune responses 6and reduce chronic inflammation in immune-mediated diseases. In recent years, the threat
20、of disease to the public is increasing, the phenomenon not only in industrialized countries but also in developed countries. In developed countries, obesity increases the risk of type 2 diabetes, cardiovascular disease, and even cancer 7,8. Obesity and long-term, low degree of inflammation related,
21、can cause insulin resistance and systemic metabolic disorders 9.With the deepening of the research and recognition on metabolic diseases, some metabolic disorders such as type 2 diabetes are regarded as the inflammatory disease. Recently, the relationship between infection and metabolic diseases has
22、 been paid more and attention. Schistosomes are a kind of important helminthes, widely distributed in the global 76 countries and regions. Parasitic diseases, as a major public health problem in the world, are a serious threat to human health, especially for poor areas. According to the World Health
23、 Organization, more than 1 billion people worldwide suffer from a variety of parasitic diseases10.An epidemiologic study on the association of previous schistosome infection (PSI) with diabetes and metabolic syndrome in rural China showed that people with PSI had significantly lower levels of adjust
24、ed fasting blood glucose, postprandial blood glucose, glycated hemoglobin A1c, and homeostasis model assessment of insulin resistance as well as a lower prevalence of diabetes and metabolic syndrome than those without PSI, suggesting that Schistosoma japonicum infection negatively correlate with met
25、abolic syndrome7. In a new study, a high-fat diet (HFD)-induced obese mice infected with Schistosoma mansoni or injected intraperitoneally with soluble egg antigen (SEA), had improved whole-body glucose tolerance and insulin sensitivity, and increased numbers of M2 macrophages in white adipose tissu
26、e8. These studies suggest that schistosomes infection or schistosome-derived molecules might have beneficial effect on hosts metabolic homeostasis. As Schistosoma japonicum and Schistosoma mansoni have many similarities in biological and immunological characters, we speculated that Schistosoma japon
27、icum infection could improve whole-body and tissue-specific insulin sensitivity in type 2 diabetes. Liver and adipose tissues are the vital organs to regulate metabolism in the host body. It has been reported that the deregulation of some microRNAs (miRNAs), such as miR-1439, miR-103, miR-10710, miR
28、-18111 and miR-80212 etc, can contribute to the development of obesity associated insulin resistance and type 2 diabetes through altering hepatic insulin sensitivity. MiR-802 was expressed in the kidney, pancreas, skeletal muscle and white fat organs, especially in the liver. Studies have shown that
29、 miR-802 is highly expressed in the liver in type 2 diabetes and reduces the sensitivity of hepatic insulin by inhibiting the expression of hepatocyte nuclear factor 1b (Hnf1b) 47; and miR-802 can occur in peripheral blood, as a biomarker for the diagnosis of type 2 diabetes 48. In our previous stud
30、y, we found that hepatic miR-802 expression was obviously decreased with the progress of Schistosoma japonicum infection in mice. In this study, mice were infected with Schistosoma japonicum and miR-802 was screened out by microRNA expression microarray. The expression of miR-802 and its downstream
31、target gene Hnf1b was studied after the infection of Schistosoma japonicum. The study also contained the effects of Schistosoma japonicum Infection on Insulin Sensitivity in Diabetic Mice (Leprdb / db). This study will deepen the understanding of the metabolism of Schistosoma japonicum infection, an
32、d also for the rational use of schistosomiasis immunology of the basic theory of prevention and treatment of related diseases provide an important reference. Materials and methods animal： schistosoma japonica positive Oncomelania： Purchased from Jiangsu Institute of parasitic diseases. mice :Thirty
33、female 6 weeks old clean grade BALB / c mice purchased from animal core facility of Nanjing Medical University were randomly divided into two groups: control group (BALB / c-con) and acute infection group (BALB / c-inf), each mouse infected 40 schistosomiasis cercariae， each group had 15 mice. Selec
34、t the dynamic Schistosoma japonicum positive Oncomelania placed in a small beaker with water without chlorine.At 25 , under normal light these Oncomelania escape cercariae. Using the sterile ring to move the cercariae to 12mm 12mm coverslips. Under the dissection microscope, 40 mice of Schistosoma j
35、aponicum were inoculated in the normal group. Couting under the anatomical lens， except for the normal group, each mouse was infected with 40 Japanese schistosomiasis cercariae. Fixed the mouse with the mouse plate, so that the abdomen up.Removing the hair from their abdomen , and wetting the site o
36、f the skin, then covered with cercariae coverslips and keeping 20 min.Six weeks after infection,8 mice were randomly selected from the infection group and the control group, extracting RNA from the Isolated liver tissue. Results figure1.miR-802 expression is increased in mice and humans. A, qRT PCR
37、quantification of C57BL/6 mice miR-802 expression in liver. B, qRT PCR quantification of C57BL/6 mice miR-802 expression in serum. C,qRT PCR quantification of human miR-802 expression in liver . All error bars indicate mean+s.e.m.; and *P 0.05, *P 0.01. figure2.different factors that regulate miR-80
38、2 expression. A, qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by SEA（ 12ug/ul， 6h） . B, qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by IL-22（ 25ug/ul， 48h） 、 TNF-（ 100ug/ul， 48h） . All error bars indicate mean+s.e.m.; and *P 0.
39、05, *P 0.01. A B C A B figure3.manual intervention： inhibition, overexpression of miR-802. A, qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by miR-802 mimic（ 24h） . B, qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by miR-802 lenti
40、virus（ 48h） .C,qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by miR-802 inhibitor（ 24h） , All error bars indicate mean+s.e.m.; and *P 0.05, *P 0.01. figure4.the expression of the target gene of miR-802hnf1b. A, qRT PCR quantification of hnf1b expression in liver. B
41、, qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by SEA.C,qRT PCR quantification of miR-802 expression in FL83B cell after being stimulate by IL-22、 TNF-.All error bars indicate mean+s.e.m.; and *P 0.05, *P 0.01. A B C A B figure5.detection of related indicators of
42、glucose metabolism in hnf1b. A B C D A Discussion as most helminth infections in humans are chronic in nature, it would be important to test whether the also occurs in a model of chronic infection. Chronic inflammation associated with obesity contributes to some metabolic syndromes, such as insulin
43、resistance and type 2 diabetes. The type 2 immune responses induced by Schistosoma mansoni infection and egg antigens immunization were reported to improve the, and tissue-specific insulin sensitivity of adipose tissue in obese mice. In the 20th century, great effort was put into the worldwide contr
44、ol of infectious diseases. However, the decrease in parasitic and other infectious diseases was associated with a substantial increase in prevalence of chronic inflammatory disorders such as asthma, autoimmune diseases (type 1 diabetes, multiple sclerosis), and inflammatory bowel disease.5 Although
45、the prevalence of asthma and allergic disorders seems to have stabilised in developed countries, the prevalence has increased in developing countries.6 These epidemiological results accord with the hygiene hypothesis or derivatives, such as the so-called old friends hypothesis7 and the biodiversity
46、hypothesis,8 which suggest that the removal of the regulatory effects of microorganisms and parasitesfrom populations genetically adapted to live with themtends to lead to an imbalance in the immune system and an increase in immune-mediated diseases.9 Consequently, the question arises of whether hel
47、minthes should be regarded as harmful pathogens or as beneficial commensals. In low-resource settings deworming is advocated to prevent worm-associated morbidity,10 whereas several research groups in high-income countries are investigating the therapeutic potential of worms and their secreted produc
48、ts in the treatment of inflammatory diseases. This paradox needs to be carefully considered because the practical implications are manifold. THE OBESITY EPIDEMIC REPRESENTS A growing threat to public health, not only in industrialized countries but also in urban centers of developing countries. Obes
49、ity significantly increases the risk for the development of type 2 diabetes, cardiovascular diseases, and eventually, cancer (1, 2) and is often associated with a state of chronic, low-grade inflammation, which contributes to tissue-specific insulin resistance and whole-body metabolic dysfunction (3). Among the underlying molecular mechanisms, classically activated (M1) macrophages were shown to accumulate in WAT from obese mice, where t