Octreotide modulates the expression of somatostatin receptor subtypes in inflamed rat jejunum induced by Cryptosporidium parvum

Abstract

Somatostatins are proteins that are involved in gastrointestinal function. However, little is known with regard to somatostatin receptor subtype (SSTR) expression changes that occur in the jejunum during low-grade inflammation and during subsequent octreotide treatment. The aim of the present study was to investigate the expression of SSTRs in the jejunums of Cryptosporidium parvum (C. parvum)-infected rats by immunohistochemisty, reverse transcription (RT) PCR and quantitative real-time RT-PCR assays. Five-day-old suckling Sprague-Dawley rats (n = 15 for each group) were orally gavaged with 105 Nouzilly isolate (NoI) oocysts. Rats then received 50 μg/kg/day of octreotide by intraperitoneal injection from day 10 to day 17 post-infection. Animals were sacrificed on days 7 and 14 post-infection for immunohistochemical analysis and on days 14, 35 and 50 for mRNA expression analysis of SSTR subtypes. Histological analysis of jejunum tissues demonstrated infection of C. parvum along the villus brush border on day 7 post-infection and infection clearance by day 14 post-infection. Real-time PCR analysis indicated that in the inflamed jejunum, a significant increase in SSTR1 and SSTR2 expression was observed on day 14 post-infection. Octreotide therapy down-regulated the expression of SSTR2 on day 37 post-infection but significantly increased expression of SSTR1, SSTR2 and SSTR3 on day 50 post-infection. The results indicate that specific SSTRs may regulate the inflammatory pathway in the rat intestinal inflammation model.

Fig 1. Representative immunohistochemical images of alterations in jejunal, ileal and colonic mucosae following C. parvum infection.

A, B and C represent jejunum, ileum and colon tissues, respectively, following 7 days ofinfection. The red arrows indicate typical parasite formations. D, E and F represent jejunum, ileum and colon tissues, respectively, following 14 days of infection. Magnification x 400. C. parvum, Cryptosporidium parvum.

Fig 2. Parasite formation counts in jejunal and ileal mucosae from C. parvum-infected animals.

Four-micrometer sections were stained with Giemsa and considered infected if at least one cryptosporidial developmental form was observed within one mucosal cell, as determined previously [13]. At least 20 fields were counted that contained the highest number of parasites. The values represent the mean ± standard deviation (n = 5 in each group). C. parvum, Cryptosporidium parvum.

Fig 3. Representative gel agarose images of SSTR PCR products following q-PCR analysis.

A, A 2% agarose gel was stained with ethidium bromide following qPCR to verify the detection of the gene products. (MW) 100 bp DNA ladder, (1) brain SSTR1, 54 bp, (2) jejunum SSTR1, 54 bp, (3) brain SSTR2, 107 bp, (4) jejunum SSTR2, 107, bp, (5) jejunum SSTR3, 111 bp. B, A 1.8% agarose gel was stained with ethidium bromide following qPCR to verify the detection of the gene products. (MW) 50 bp DNA ladder, (1) jejunum β-actin, 52 bp, (2) brain β-actin, 52 bp, (3) jejunum SSTR1, 54 bp, (4) brain SSTR1, 54 bp. Black arrows indicate the 100 bp marker.

Fig 4. Sequential modulation of SSTR1, SSTR2 and SSTR3 jejunal mRNA levels in C. parvum-infected or octreotide-treated rats on day 14 post-infection.

The bar chart represents the quantification of the PCR results that corresponds to the mRNA levels of expression of SSTR genes. The values represent mean ± standard deviation (n = 5 in each group). *P<0.05, **P<0.01, infected, untreated vs. control and infected, untreated vs. infected, treated. C. parvum, Cryptosporidium parvum.

Fig 5. Sequential modulation of SSTR1, SSTR2 and SSTR3 jejunal mRNA levels in C. parvum-infected or octreotide-treated rats on A. day 37 post-infection B. day 50 post-infection.

The bar charts represent the quantification of the PCR results that correspond to the mRNA levels of expression of SSTR genes. The values represent mean ± standard deviation (n = 5 in each group). *P<0.05, **P<0.01, infected, untreated vs. control and infected, untreated vs infected, treated. C. parvum, Cryptosporidium parvum.

Fig 6. PGP 9,5 immunoreactivity in rat jejunum infected by C.parvum on days 14 and 37 post-infection.

Protein gene product 9.5 (PGP 9.5) is a marker for neuroendocrine cells. The immunohistochemistry of routinely processed neuronal tissues was conducted in tissues that were obtained at days 14 and 37 for the 4 different groups, namely infected, control, infected untreated and infected treated.