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theses

Lactobacillus rhamnosus GG (LGG) is a well studied and commercially available probiotic, known to be able to alleviate intestinal disorders. From previous research in Dr. Ferraris’s lab, we discovered that by direct perfusion of active LGG to the jejunum of the small intestine, we were able to promote the production of anti-inflammatory cytokines IFN-γ, IL10, IL2, IL4, IL5, and IL6 (P<0.05) in four hours. To further study the properties of the LGG perfusate, we conducted a feeding study, where we gavage fed Mus Musculus (30 mice, 22.3±2 g Body Weight) with either Saline or LGG (6.6 x 108 CFU/mL in Saline) two times a day for days, then perfused with bacterial LPS or Poly I:C to the small intestine to determine whether prior consumption of LGG prevented bacterial or viral induced inflammation (n=5). Unfortunately, the results generated were statistically insignificant. Suggesting that gavage feeding LGG may have failed to stimulate an intestinal reaction, probably because of stress and dilution with endogenous bacterial. We performed another perfusion experiment followed by transcriptional analysis of the small intestinal flora. LGG and Saline perfused mice results of the RNAseq were analyzed by IPA (Ingenutiy Pathway Analysis), a software that is able to predict the activities of molecular pathways. Among pathways predicted by IPA to be affected by LGG perfusion, “LPS/IL-1 Mediated Inhibition of RXR (Retinoid X receptor) Function” has the most significant P value. Hence, using IPA’s result as a map, I determined to focused my attention towards LGG’s effect on this pathway. The “LPS/IL-1 Mediated Inhibition of RXR Function” pathway is a Mitogen-activated protein kinase (MAPK) pathway, with JNK as the MAPK. Bacterial LPS would activate JNK by phosphorylation. The pJNK would in turn inhibits RXR activation, thus inhibiting the production of its’ downstream associate enzymes and transporter. According to our RNAseq data, IPA predicted the pathway inhibited with LGG perfusion. Meaning that LGG would decrease pJNK, resulting in an increase in downstream transcription. There are a total of 25 effectors (enzymes and transporters) associated with xenobiotic metabolism downstream of RXRα. Among the effectors, the mRNA levels of 9 (CYP2A6, CYP2C8, CYP2C9, CYP3A5, CYP3A7, ALDH, PAPSS2, SULT, GST) were measured in previous RNAseq experiment, 12 (MGMT, SOD3, FMO2, UGT, MRP2, MRP3, MRP4, OATP2, CES, MAOA, CAT, MDR1) were measured by qPCR. Two receptors within the RXR complex (CAR, PXR), a known downstream substrate of JNK (c-JUN), and an upstream receptor of the pathway were also measured by qPCR. The phosphorylation of JNK and EGFR were detected and analyzed with western bloting. From RNAseq, the mRNA expression of 9 effectors (CYP2A6, CYP2C8, CYP2C9, CYP3A5, CYP3A7, ALDH, PAPSS2, SULT, GST) showed an increase of >1.5 fold with LGG perfusion compared to Saline Perfusion (P<0.05). From our qPCR results, downstream of RXR, when compared with saline perfused samples, the mRNA expression of 10 effectors (SOD3, FMO2, UGT, MRP2, MRP3, MRP4, OATP2, CES, MAOA, MDR1) increased more than 1.2 fold with LGG perfusion (P<0.05). The phosphorylation of JNK decreased in LGG perfused samples when compared with the control. In conclusion, we have discovered strong evidence showing that LGG has the ability to increase xenobiotic metabolism in the small intestine through the inhibition of the “LPS/IL-1 Mediated Inhibition of RXR Function” pathway. More experiments are still needed to further prove this theory. However, EGFR (Epidermal growth factor receptor), increased in phosphorylation in LGG perfused samples when compared with the control. EGFR is known to be associated with the production of cytokines, hence this can be used for future experiments in explaining the reaction observed with LGG perfusion.

ETD_8280

M.S.

Includes bibliographical references

by Bowen Yang

doi:10.7282/T39K4F9P

ETD graduate

The author owns the copyright to this work.