Research code: IR.IAU.CTB.REC.1401.127
Ethics code: IR.IAU.CTB.REC.1401.127
Clinical trials code: IR.IAU.CTB.REC.1401.127
Ghazaleh Sharifi Ardani 
, Javad Arasteh * , Amir Mirzaie , Ghazaleh Lorestanizade , Hamidreza Mehrjo
, Javad Arasteh * , Amir Mirzaie , Ghazaleh Lorestanizade , Hamidreza Mehrjo
, J.arasteh@iauctb.ac.ir
Abstract: (86 Views)
Background and Aim: Ulcerative colitis is an acute inflammation condition when sudden and severe inflammation affects the inner lining of the colon and rectum (1). The clinical presentation of this condition includes stomach discomfort, diarrhea, frequent passage of bloody stools, and the development of fibrosis (3). Various therapeutic approaches have been suggested to manage this complication, including the utilization of 5-aminosalicylates (5-ASA), employed for the treatment of inflammatory bowel disease (IBD). Furthermore, immunomodulators, such as azathioprine, 6-mercaptopurine, and antibiotics, play a crucial role in the treatment of conditions that do not respond to steroids or require ongoing steroid use. Nevertheless, these medications have adverse reactions and restricted efficacy in clinical practice, prompting the quest for natural and exceptionally efficient approaches and resources (4). The gut microbiota contributes critically to gut function regulation and can directly influence human health. This finding has attracted attention to probiotic bacteria, which can modify the indigenous microbial community or intestinal flora to promote gut well-being by eradicating pathogenic microorganisms when ingested in adequate quantities. Consequently, there has been a substantial rise in interest in probiotic applications as an alternate approach to medication therapy in recent years (6). Probiotics have recently been employed in IBD management. Giunchetti et al. demonstrated that a combination of eight probiotic bacteria could effectively inhibit chronic voiding (7). Several investigations on various strains of L. plantarum have exhibited a notable reduction in the production of pro-inflammatory cytokines (TNF-α and IL-10), resulting in beneficial effects for ulcerative colitis treatment both in vitro and in vivo. They regulate the composition of the intestinal microbiota, hinder the programmed cell death of intestinal cells, and prevent the initiation of the signaling pathway (NF-kB) (13). Nano-scale-designed carriers can effectively traverse biological barriers to ensure medication safety and provide the most efficient dosage. Niosomes are uncharged lipid vesicles created by a non-ionic solvent hydration with cholesterol and other lipids. Their vesicle system can potentially serve as a carrier for lipophilic and amphiphilic drugs. The non-ionic property of the substance reduces its toxicity and prevents it from affecting cells, resulting in a higher therapeutic index for the enclosed medication (15). Hence, the present research aimed to examine the regulatory impact of Lactobacillus plantarum, encapsulated in Niosomes, on the acetic acid-induced colitis rat model by using RT-PCR. The study has primarily employed Niosomes as a carrier for probiotics in colitis treatment.
Method: This study prepared L. plantarum encapsulated in Niosomes by thin-layer hydration, focusing on physical and chemical profile assessment of nanoparticles, including their FTIR, zeta potential, size, shape, and encapsulation percentage. In addition, the level of toxicity in the normal cell line was assessed using the MTT technique. Experiments were conducted on 16 laboratory mice, randomly allocated into four groups (n=4), one group receiving L.plantarum encapsulased in Niosomes at a dose of 108 cfu/ml, one group receiving L.plantarum at a dose of 108 cfu/ml, a healthy group, and a colitis group. The rats in different treatment groups were euthanized with an overdose of ether on the specified day. Subsequently, 8 cm of colon tissue was removed, longitudinally opened and rinsed with normal saline. Various colon segments were fixed in a 10% formalin solution for histological analysis and then embedded in paraffin blocks for sectioning. Every specimen was treated with hematoxylin and eosin dye. Each group's slides were analyzed. The stained sections were assessed based on the extent of tissue injury and inflammation. The assessment of damage and inflammation involved four specific parameters: epithelial tissue damage, crypt damage, reduction in the number of goblet cells, and augmentation of inflammatory cells. After the extraction of RNA from colon tissue and subsequent cDNA synthesis, gene expression was assessed using real-time PCRa, quantified by one-way ANOVA technique, and analyzed using Rest software. A gene expression graph was generated using the PadGraph program. A significance level of P<0.05 was considered to determine statistical significance.
Result: The findings indicate that Niosome contains L.plantarum with a microencapsulation efficiency of %80, an average size of 1508.5 nm, a dispersion index of 2.44, and a positive surface charge of zeta potential of 37.8. FTIR and SEM studies revealed no interaction between L.plantarum and Niosome. Instead, the particles formed agglomerates and adhered to one another, resulting in a non-smooth surface. The particles exhibited a sphere-like morphology with rough edges. Additionally, cellular tests revealed that the toxicity of the encapsulated form was greater than that of the non-encapsulated form. The administration of L.plantarum encapsulated with Niosome resulted in a notable decrease (p<0.005) in macroscopic symptoms. Microscopic examination of the L.plantarum group, when contained by Niosomes, revealed the presence of inflammation and minimal damage to the epithelial cells. The results exhibited a substantial divergence from the colitis group. The administration of encapsulated L. plantarum substantially decreased the production of TNF-α (P<0.05) compared to the colitis group.
Discussion: This study aimed to investigate the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 expression levels in the colon tissue of male rats with colitis. The rats were treated with L.plantarum and L.plantarum encapsulated in Niosome at a concentration of colony-forming units per milliliter (cfu/ml). L.plantarum, when encapsulated, exhibited a substantial effect on the cytokines IL-10 and TNF-α, causing a drop in the former and a rise in the latter. Nevertheless, there was no evidence regarding the efficacy of the nanocarrier as a viable vehicle for acute ulcerative colitis treatment in rats. The patient group without therapy had a significant reduction in epithelial cells, an increase in inflammatory cells, a decrease in goblet cells, and high levels of crypt abnormalities. The parameters in the groups treated with L.plantarum and encapsulated with L.plantarum declined considerably. In a 2022 publication, Israr Khan et al. demonstrated that L.plantarum administration to ulcerative colitis colon cells effectively ameliorated colitis produced by dextran sulfate (DSS) in animals. The positive effects of consuming these bacteria were evident through increased body weight, food and water consumption, and colon length Additionally, there was a decrease in disease activity index (DAI), inflammatory variables, and histological scores (19). Research conducted by Duary et al. showed that L.plantarum Lp substantially reduced the expression of TNF-α and COX2 genes by 0.026 and 0.077, respectively, in a mouse model with colitis. Meanwhile, the expression level of 10-IL was considerably upregulated by 813.37 and 337.1 times in mice with colitis and those without colitis treated with plantarum Lp91 (24).
Method: This study prepared L. plantarum encapsulated in Niosomes by thin-layer hydration, focusing on physical and chemical profile assessment of nanoparticles, including their FTIR, zeta potential, size, shape, and encapsulation percentage. In addition, the level of toxicity in the normal cell line was assessed using the MTT technique. Experiments were conducted on 16 laboratory mice, randomly allocated into four groups (n=4), one group receiving L.plantarum encapsulased in Niosomes at a dose of 108 cfu/ml, one group receiving L.plantarum at a dose of 108 cfu/ml, a healthy group, and a colitis group. The rats in different treatment groups were euthanized with an overdose of ether on the specified day. Subsequently, 8 cm of colon tissue was removed, longitudinally opened and rinsed with normal saline. Various colon segments were fixed in a 10% formalin solution for histological analysis and then embedded in paraffin blocks for sectioning. Every specimen was treated with hematoxylin and eosin dye. Each group's slides were analyzed. The stained sections were assessed based on the extent of tissue injury and inflammation. The assessment of damage and inflammation involved four specific parameters: epithelial tissue damage, crypt damage, reduction in the number of goblet cells, and augmentation of inflammatory cells. After the extraction of RNA from colon tissue and subsequent cDNA synthesis, gene expression was assessed using real-time PCRa, quantified by one-way ANOVA technique, and analyzed using Rest software. A gene expression graph was generated using the PadGraph program. A significance level of P<0.05 was considered to determine statistical significance.
Result: The findings indicate that Niosome contains L.plantarum with a microencapsulation efficiency of %80, an average size of 1508.5 nm, a dispersion index of 2.44, and a positive surface charge of zeta potential of 37.8. FTIR and SEM studies revealed no interaction between L.plantarum and Niosome. Instead, the particles formed agglomerates and adhered to one another, resulting in a non-smooth surface. The particles exhibited a sphere-like morphology with rough edges. Additionally, cellular tests revealed that the toxicity of the encapsulated form was greater than that of the non-encapsulated form. The administration of L.plantarum encapsulated with Niosome resulted in a notable decrease (p<0.005) in macroscopic symptoms. Microscopic examination of the L.plantarum group, when contained by Niosomes, revealed the presence of inflammation and minimal damage to the epithelial cells. The results exhibited a substantial divergence from the colitis group. The administration of encapsulated L. plantarum substantially decreased the production of TNF-α (P<0.05) compared to the colitis group.
Discussion: This study aimed to investigate the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 expression levels in the colon tissue of male rats with colitis. The rats were treated with L.plantarum and L.plantarum encapsulated in Niosome at a concentration of colony-forming units per milliliter (cfu/ml). L.plantarum, when encapsulated, exhibited a substantial effect on the cytokines IL-10 and TNF-α, causing a drop in the former and a rise in the latter. Nevertheless, there was no evidence regarding the efficacy of the nanocarrier as a viable vehicle for acute ulcerative colitis treatment in rats. The patient group without therapy had a significant reduction in epithelial cells, an increase in inflammatory cells, a decrease in goblet cells, and high levels of crypt abnormalities. The parameters in the groups treated with L.plantarum and encapsulated with L.plantarum declined considerably. In a 2022 publication, Israr Khan et al. demonstrated that L.plantarum administration to ulcerative colitis colon cells effectively ameliorated colitis produced by dextran sulfate (DSS) in animals. The positive effects of consuming these bacteria were evident through increased body weight, food and water consumption, and colon length Additionally, there was a decrease in disease activity index (DAI), inflammatory variables, and histological scores (19). Research conducted by Duary et al. showed that L.plantarum Lp substantially reduced the expression of TNF-α and COX2 genes by 0.026 and 0.077, respectively, in a mouse model with colitis. Meanwhile, the expression level of 10-IL was considerably upregulated by 813.37 and 337.1 times in mice with colitis and those without colitis treated with plantarum Lp91 (24).
Type of Study: Research |
Subject:
Microbiology
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