Best of the Best Gen Surg - Therapeutic Effect of Oridonin against Inflammatory Bowel Disease Involves Inhibition of Intestinal Fibrosis via NF-kB and p38STAT1 Pathways - Dr. Mao

So we're gonna go on to the second paper from SSAT, and this is Doctor Ru Min Mao, and the paper is entitled Therapeutic Effects of Ridinin Against Inflammatory Bowel Disease Involves inhibition of Intestinal fibrosis via NFKB and P38/STAT1. And uh please present the paper. So, hello, everyone. My name is Diana Mao. I'm a resident at the University of Texas Medical Branch. I would like to thank GlobalC MD and the SSAT for the opportunity to share our project. The therapeutic effect of oroonin against inflammatory bowel disease involves inhibition of intestinal fibrosis via NF kappa B and P38 1 pathways. I have nothing to disclose. So inflammatory bowel disease or IBD affects approximately 3 million adults in the United States, and it has significant costs for both patients and society. Annual medical expenses for a patient with IBD is estimated to be $15,000 and the value of work-related opportunity lost has been reported to be up to $5.5 billion. Intestinal fibrosis is a common sequela of IBD. It's found more frequently in Crohn's, affecting 30-50% of those patients, but it is also seen with ulcerative colitis, although less frequently at a rate of 5%. The fibrosis process can lead to complications that present clinically in the form of strictures, obstruction, and fistulas. EMT or epithelial mesenchymal transition is a process that contributes to the development of fibrosis. To elaborate further on EMT, it is a process that occurs on the cellular level in response to inflammation such as that present in IBD. It's defined by the loss of adherences, tight junctions, and cytoskeleton proteins that are normally found in epithelial cells. So epithelial markers such as EC adherin and ZO1 or zona occludins one. are lost. These cells, in fact, gain extracellular matrix and fibroblast morphology as well as migratory capacity. Fibroblast markers such as alpha-SMA or alpha smooth muscle actin and fibrinectin also are increasingly expressed. Consequently, the intestinal barrier is compromised, and this damage results in greater permeability through the intestinal wall. Our compound of interest is oroonin, a diterpenoid that's derived from the plant species Rhabdosa rubiscens. This plant has been used for hundreds of years in eastern medicine to treat common ailments such as cough, abdominal pain, and muscle aches. Research has demonstrated that this compound also has antioxidant, anti-tumor, and anti-inflammatory effects on various tissues. And it's thought that these effects are due to the inhibition of multiple signaling pathways including NFKpa B and P38. In the context of the gastrointestinal tract and IVD, oroonin has also been described to have therapeutic effects. In studies with in vitro experiments in animal models, oroonin has resulted in the slower disease progression and decreased levels of inflammatory cytokines and cells. However, the molecular mechanism by which this occurs remains largely unknown. We had two hypotheses with our study. First, we hypothesized that oroonin would attenuate inflammation-related EMT and fibrogenesis, and second, we hypothesized that this would occur through suppression of known pro-inflammatory pathways such as NF kappa B and P38. To conduct our study, we used rat intestinal epithelial cells and the cell line was IEC-6. We pre-treated these cells with oroonin and then exposed them to LPS or lipopolysaccharide to trigger inflammation that was representative of that seen in IBD. Following this, Western blots and immunofluorescence assays were used to analyze specific proteins. Our results first demonstrated that oroonin inhibited LPS induced fibrosis markers and restored ZO1 expression, which supports our first hypothesis. The western blot and its densitometric analysis on the right show that exposure to LPS increases levels of alpha-SMA, which is a marker of EMT and fibrosis. However, this effect was inhibited by pre-treatment with oroonin. The same phenomenon was observed with fibrinectin, which is another marker of EMT and fibrosis. However, the opposite effect was seen with CO1, which is a tight junction protein and normally highly expressed in epithelial cells. Exposure to LPS decreased CO1 levels, and pre-treatment oroonin was able to attenuate that effect and restore ZO1 expression. Our second finding was that oroonin inhibited LPS induced NF kappa B activation. Prior research has demonstrated that NF kappa B is a key pro-inflammatory pathway that regulates alpha-SMA, which is how it ties into our study. So oroonin inhibited the activation of NF kappa B by preventing the nuclear translocation of NF kappa BP65, which is a representative subunit. This was seen with immunofluorescent staining, and with LPS you can see that P65 has translocated to the cell nucleus. However, with Lorido in pre-treatment, this effect is diminished and P65 has a greater distribution within the cytoplasm. The same effect is seen on the western blot, and on the two right columns, you can appreciate that following oroonin exposure, the signal of the nuclear components of P65 has been attenuated. We also involve TNF alpha on these western blocks because it's a pro-inflammatory compound with known involvement in IBD and the NF kappa B pathway. Orodonins inhibition alpha provides further support of its anti-inflammatory role within IBD. In addition to its effects on P65, we also found that oroonin inhibited the phosphorylation of IKK alpha beta and I kappa B alpha, and prevented total I kappa B alpha degradation. As a brief overview, NF kappa B is normally inhibited when bound by I kappa B alpha. The phosphorylated IKK alpha beta in turn phosphorylatesi kappa B alpha, which releases NF kappa B and allows it to translocate to the mucus. Phosphorylated kappa alpha then undergoes degradation. This western blot demonstrates that following the administration of oroonin, there is a decrease in the phosphorylation of IKK alpha beta and I Kappa B alpha. Inhibiting the phosphorylation of I kappa B alpha also increases the total expression and levels of Ikappa B alpha, so that it's it's, it's expression is greater with oroonin. Our third finding was that oroonin inhibited the LPS induced P38 and statin pathway as well. This required a two-part experiment, the first of which involved the specific P38 inhibitor, SB 203,580. This P38 inhibitor dose dependently attenuated the LPS induced alpha-SMA expression, as seen on both the Western blood and densometric analysis. Oronin is involved because it inhibits LPS induced stat 1 activity, which is a downstream factor of P38. Again, this is seen in both the Western blot and density metric analysis. In conclusion, we found that orodonin inhibits EMT-related fibrosis and does so via inhibition of the NF kappa B and P38 1 pathways. Consequently, oroonin may be a promising drug candidate for IBD and specifically within the context of fibrosis. Thank you for your time and attention. This is my email address here. I'm happy to hear any emails and answer any questions that you all may have. Thank you so much. Thank you, Diana. I'd just like to ask you, where did you find oroonin? What is it? Where did you get it? Yes, so it actually is derived from, you know, China. It's used a lot in their eastern medicine and has had therapeutic effects on common ailments and been used for 100. Hundreds of years. And so I guess, um, in the Chinese literature, there's been a lot of interest as to why this has been utilized and why it's been, you know, effective. And so after breaking down the actual plant, they found that the most active compound was oroonin. And from there, that sort of expanded to utility and other realms and then on other, you know, disease processes and eventually found its way to us, you know, with our IBD and, and GI research. Well, good, we're going back to basics again, I think, you know, foxglove plant type stuff. Todd, do you have a question? Well, I think it points out a bigger a bigger point here in that, you know, uh, my mother-in-law, uh, is Chinese and always gives me, uh, giving me Chinese, um, medicines in our family, and we sometimes roll our eyes, and, you know, they would seem to work. If the question is we just haven't studied them. So, you know, one at a time, we're going to have to do this. We're going to have to take. These therapies, these holistic therapies that people have, uh, you know, claim work, and this is a big, big, uh, point, this is a big discovery for that reason. Not only is, could this be great for IBD, um, but we know people who have gone to get, uh, alternative medicine for IBD and they claim it works better than any of the other, um, you know, medicines we have. So, I think this is phenomenal work. I understood about 1% of it, but, uh, but it, it was, uh, it was, I mean, it's a big deal. So, as someone who had IBD. I'm excited to see that we really don't have great therapies yet, so that's great. All right, we're going to try to catch up with Dr. Rosen. Do you have any pressing comments here? I thought it was great. I, I agree with everything. Sometimes the answer isn't as complex as we think, so good for you for looking into it. You can use it on your hernia patients. I'm sure every one of them is going to get. I try everything to not operate on those people. OK very good thank you so much Diana thank you so we're going to now go to a poll for the uh SSAT papers they were great papers and uh let me just uh read them to you and and we'll have the poll um the the first. Paper by Ryan Morgan was development of postoperative local tumors and distant metastasis relative to a low fat diet, and the second paper by Doctor Mao Diana Mao was therapeutic Effective orodonin. So please vote. And uh we'll have your results of the vote in just a moment and the vote's moving back and forth, so please vote now. Uh, the vote is going back and forth. It uh it just got tied uh so keep going and uh and it it's moving a lot.