Date of Award

4-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

First Advisor

Dr. Sandeep B. Subramanya

Second Advisor

Dr. Shreesh K. Ojha

Abstract

Inflammatory bowel diseases, which comprise Crohn’s disease (CD) and ulcerative colitis (UC), are chronic and progressive immune-mediated inflammatory conditions of the gastrointestinal (GI) tract. Both genetic and environmental factors influence this condition. Conventional therapy to suppress aberrant immune responses seen in IBD with corticosteroid or with biological agents has a high relapse rate that limits their use. Therefore, approximately 40% of IBD patients switch to alternative therapies that include dietary supplements rich in phytochemicals. Nerolidol (NED) is a naturally occurring sesquiterpene alcohol present in various plants and has potent anti-inflammatory properties. Therefore, in the current study, the role of NED in preclinical models of colon inflammation were investigated. Initial experiments were carried out to investigate the anti-inflammatory properties of NED in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. NED significantly decreased proinflammatory cytokine such as TNF-α, IL-1β, and IL-6 both at protein and mRNA level in LPS-stimulated RAW macrophages. In addition, NED also significantly decreased expression of proinflammatory mediators such as COX-2 and iNOS. Based on these results NED was further investigated as a putative anti-inflammatory compound in in vivo and in vitro models of colon inflammation. C57BL/6J male black mice administered with 3% dextran sodium sulfate (DSS) used as in vivo model of colon inflammation. DSS-induced colitis groups received either vehicle or NED (50, 100, and 150 mg/kg body weight/per day) by oral gavage. NED significantly decreased the DAI, colon length and preserved microscopic architecture of the colon to near control level. NED significantly decreased tissue MPO concentrations, CXCL-2, CCL2 mRNA expression and proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and mediators (COX-2 and iNOS) level both at the protein and at mRNA level in DSS administered mice. NED promoted Nrf-2 nuclear translocation and increased antioxidant enzyme (SOD and CAT) activity, HO-1 and SOD-3 mRNA levels. In vitro model of colon inflammation was established by challenging HT-29 cells with TNF-α (1ng/ml concentration) and treated with NED (25μM and 50μM). NED treatment significantly decreased proinflammatory chemokines (CXCL-1, IL-8, CCL2, and COX-2) mRNA levels.

NED significantly decreased phosphorylation of MAPK (p38, JNK, and ERK1/2) and NF-κB in the DSS-induced colitis and in LPS-stimulated RAW macrophages.

Intestinal epithelial barrier dysfunction leading to enhanced intestine permeability is associated with IBD pathogenesis. Therefore, the effect of NED on intestine tight junction integrity using DSS-induced colitis and in LPS challenged Caco-2 monolayers was investigated. NED significantly decreased FITC-dextran permeability in DSS group and decreased transepithelial electrical resistance (TEER) in LPS-treated Caco-2 monolayers. NED significantly increased expression of tight junction proteins such as claudin-1, -3, -7, and occludin in in vitro and in vivo models. Collectively, results from the present study indicates that NED supplementation attenuates colon inflammation through its potent antioxidant and anti-inflammatory activity and enhances intestine tight junction integrity.

Arabic Abstract

أمراض اﻷﻣﻌﺎء اﻻﻟﺘﮭﺎﺑﯿﺔ، واﻟﺘﻲ ﺗﺸﻤﻞ ﻣﺮض ﻛﺮون وﻣﺮض اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن اﻟﺘﻘﺮﺣﻲ، ﺗﻌﺪ أﻣﺮاض اﻟﺘﮭﺎﺑﯿﺔ ﻣﺰﻣﻨﺔ وﻣﺴﺘﻔﺤﻠﺔ ﺗﺼﯿﺐ اﻟﺠﮭﺎز اﻟﮭﻀﻤﻲ. ﺗﻠﻌﺐ اﻟﻌﻮاﻣﻞ اﻟﻮراﺛﯿﺔ واﻟﺒﯿﺌﯿﺔ دورا ﻓﻲ اﻹﺻﺎﺑﺔ ﺑﮭﺬه اﻷﻣﺮاض. اﻟﻜﻮرﺗﯿﻜﻮﺳﺘﯿﺮوﯾﺪ أو اﻟﻌﻮاﻣﻞ اﻟﺒﯿﻮﻟﻮﺟﯿﺔ اﻟﻤﺴﺘﺨﺪﻣﺔ ﻓﻲ اﻟﻌﻼج اﻟﺘﻘﻠﯿﺪي ﻟﻘﻤﻊ اﻟﻤﻨﺎﻋﺔ اﻟﺸﺎذة ﻓﻲ ﻣﺮض اﻟﺘﮭﺎب اﻷﻣﻌﺎء ﺗﻌﺪ ذات اﻧﺘﻜﺎس ﻣﺮﺗﻔﻊ، ﻣﻤﺎ ﯾﺆدي ﻟﻠﺤﺪ ﻣﻦ اﺳﺘﺨﺪاﻣﮭﺎ. ﯾﻠﺠﺄ ﻣﺎ ﯾﻘﺮب ﻣﻦ 40٪ ﻣﻦ ﻣﺮﺿﻰ داء اﻷﻣﻌﺎء اﻻﻟﺘﮭﺎﺑﻲ إﻟﻰ اﻟﻌﻼﺟﺎت اﻟﺒﺪﯾﻠﺔ واﻟﺘﻲ ﺗﺸﻤﻞ اﻟﻤﻜﻤﻼت اﻟﻐﺬاﺋﯿﺔ اﻟﻐﻨﯿﺔ ﺑﺎﻟﻤﻮاد اﻟﻜﯿﻤﯿﺎﺋﯿﺔ اﻟﻨﺒﺎﺗﯿﺔ.

ﻧﯿﺮوﻟﯿﺪول ﻋﺒﺎرة ﻋﻦ ﻛﺤﻮل ﺳﯿﺴﻜﯿﺘﯿﺮﺑﯿﻦ طﺒﯿﻌﻲ ﻣﻮﺟﻮد ﻓﻲ ﻧﺒﺎﺗﺎت ﻣﺨﺘﻠﻔﺔ ذات ﺧﺼﺎﺋﺺ ﻗﻮﯾﺔ ﻣﻀﺎدة ﻟﻼﻟﺘﮭﺎب. ﻓﻲ اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ، ﻗﻤﻨﺎ ﺑﺘﺤﺮي دور ﻧﯿﺮوﻟﯿﺪول ﻓﻲ اﻟﻨﻤﺎذج ﻗﺒﻞ اﻟﺴﺮﯾﺮﯾﺔ ﻻﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن. ﻓﻲ ﺗﺠﺎرﺑﻨﺎ اﻟﻤﺒﺪﺋﯿﺔ ﻗﻤﻨﺎ ﺑﺪراﺳﺔ اﻟﺨﺼﺎﺋﺺ اﻟﻤﻀﺎدة ﻟﻼﻟﺘﮭﺎﺑﺎت ﻟـﻨﯿﺮوﻟﯿﺪول ﻓﻲ ﺧﻼﯾﺎ اﻟﺒﻼﻋﻢ اﻟﻤﺤﻔﺰة ﻋﺪﯾﺪات اﻟﺴﻜﺎرﯾﺪ اﻟﺪھﻨﯿﺔ (LPS) ﺑﺎﺳﺘﺨﺪام ﻓﺌﺮان (264.7 RAW). ﺛﺒﻂ ﻧﯿﺮوﻟﯿﺪول ﻋﺪﯾﺪات اﻟﺴﻜﺎرﯾﺪ اﻟﺪھﻨﯿﺔ اﻟﺘﻲ ﺗﻔﻌﻠﮭﺎ ﻣﻮاد ﺗﻨﺘﺞ ﻓﻲ ﻣﻮاﻗﻊ اﻻﻟﺘﮭﺎب ﻣﺜﻞ TNF-α و IL-1β و6-IL. وذﻟﻚ ﻋﻦ طﺮﯾﻖ ﺗﺤﻔﯿﺰ اﻧﺘﺎج ﺣﻤﺾ اﻟﺮﯾﺒﻮﻧﻮﻛﻠﯿﯿﻚ اﻟﺨﺎص ﺑﮭﺎ. ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ذﻟﻚ، ﻣﻨﻊ ﻧﯿﺮوﻟﯿﺪول اﻟﺰﯾﺎدة ﻓﻲ اﻟﺘﻌﺒﯿﺮ ﻋﻦ اﻟﻮﺳﺎﺋﻂ اﻟﻤﺆﯾﺪة ﻟﻼﻟﺘﮭﺎﺑﺎت ﻣﺜﻞ 2-COX وiNOS ﻣﻤﺎ ﯾﺸﯿﺮ إﻟﻰ ﺧﺼﺎﺋﺼﮫ اﻟﻤﺤﺘﻤﻠﺔ ﻛﻤﻀﺎد ﻟﻼﻟﺘﮭﺎﺑﺎت. وھﺬا اﻟﺪور ﺗﻢ دراﺳﺘﮫ ﻣﺨﺒﺮﯾﺎ ﻓﻲ ﻧﻤﺎذج ﻣﺨﺒﺮﯾﺔ ﻣﻌﺮوﻓﺔ ﻟﻤﺮض اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﻓﻲ اﻟﻔﺌﺮان واﻟﻤﺨﺒﺮ. ﺗﻢ إﻧﺸﺎء ﻧﻤﻮذج اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﺑﺎﺳﺘﺨﺪام اﻟﻔﺌﺮان اﻟﺴﻮداء اﻟﺬﻛﻮر (C57BL / 6J) 6J / C57BL اﻟﺘﻲ ﯾﺘﻢ اطﻌﺎﻣﮭﺎ 3 ٪ ﻛﺒﺮﯾﺘﺎت اﻟﺼﻮدﯾﻮم دﯾﻜﺴﺘﺮان (DSS) ﻓﻲ ﻣﯿﺎه اﻟﺸﺮب ﻟﻤﺪة 7 أﯾﺎم ﻟﻠﺤﺚ ﻋﻠﻰ اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﺑﮭﺎ. ﺧﻼل اﻟﺘﺠﺮﺑﺔ اﻟﻌﻠﻤﯿﺔ ﺗﻠﻘﺖ ﻣﺠﻤﻮﻋﺎت اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﻣﺮﻛﺐ ﻧﯿﺮوﻟﯿﺪول ﺑﺠﺮﻋﺎت ﻣﺘﻔﺎوتة (50 ، 100، 150 ﻣﻠﻐﻢ / ﻛﻐﻢ ﻣﻦ وزن اﻟﺠﺴﻢ / ﯾﻮﻣﯿًﺎ ﺑﺎﻟﺘﺰﻗﯿﻢ). ﺗﻢ ﻗﯿﺎس طﻮل اﻟﻘﻮﻟﻮن ، ﻣﺆﺷﺮ ﻧﺸﺎط اﻟﻤﺮض (DAI) ، أﻧﺴﺠﺔ اﻟﻘﻮﻟﻮن ، واﻟﻌﺪﯾﺪ ﻣﻦ اﻟﻤﻌﻠﻤﺎت اﻟﺒﯿﻮﻛﯿﻤﯿﺎﺋﯿﺔ. ﺗﻢ إﻧﺸﺎء ﻧﻤﻮذج اﻟﺘﮭﺎب ﻓﻲ اﻟﻤﺨﺘﺒﺮ ﻋﻦ طﺮﯾﻖ ﺗﺤﻔﯿﺰ ﺧﻼﯾﺎ 29-HT (ﺧﻠﯿﺔ ﺳﺮطﺎن اﻟﻘﻮﻟﻮن واﻟﻤﺴﺘﻘﯿﻢ اﻟﺒﺸﺮﯾﺔ) مع TNF-α (ﺗﺮﻛﯿﺰ 1 ﻧﺎﻧﻮﻗﺮام/ﻣﻠﻢ 1) ﺑﺎﺳﺘﺨﺪام ﺗﺮﻛﯿﺰات ﻣﺨﺘﻠﻔﺔ ﻣﻦ ﻧﯿﺮوﻟﯿﺪول (25 ﻣﯿﻜﺮوﻣﺘﺮ و 50 ﻣﯿﻜﺮوﻣﺘﺮ) ﻓﻲ اﻟﺘﺠﺎرب اﻟﻤﺨﺒﺮﯾﺔ. وﻧﺘﺞ ﻋﻨﮭﺎ ﺗﻘﻠﯿﻞ ﻛﺒﯿﺮ في وﺣﺪة اﻻﻟﺘﮭﺎب ﺑﺎﻷﻧﺴﺠﺔ اﻟﻤﻘﯿﻤﺔ ﻣﺠﮭﺮﯾﺎ وﻗﻠﻞ ﻣﻦ اﻟﺘﻘﻠﺺ ﻓﻲ طﻮل اﻟﻘﻮﻟﻮن. وﻧﺘﺞ ﻋﻦ اﺳﺘﺨﺪام ﻧﯿﺮوﻟﯿﺪول ﺗﻐﯿﺮ ﻓﻲ اﻟﺘﺮﻛﯿﺰ اﻟﺨﻠﻮي ﻟﻤﺎدة MPO ﺑﺎﻷﻧﺴﺠﺔ، واﻧﺨﻔﺎض ﺑﺤﻤﺾ اﻟﺮﯾﺒﻮﻧﻮﻛﻠﯿﯿﻚ ﻟﻤﺎدﺗﻲ CXCL-2 وCCL واﻟﺬي ﺗﻔﺮزة خلايا اﻟﻌﺪﻻت واﻟﺒﻼﻋﻢ ﺑﺎﻹﺿﺎﻓﺔ ﻻﻧﺨﻔﺎض ﻣﻠﺤﻮظ ﻟﻤﻮاد اﻟﻤﺆﯾﺪة ﻟﻼﻟﺘﮭﺎب وﻣﻨﮭﺎ TNF-α و IL-1β و6-IL ﻋﻠﻰ ﻣﺴﺘﻮى اﻧﺘﺎج ﺣﻤﺾ اﻟﺮﯾﺒﻮﻧﻮﻛﻠﯿﯿﻚ واﻟﺒﺮوﺗﯿﻦ اﻟﺨﺎص ﺑﮭﺎ.

ارﺗﻔﺎع ﻓﻲ اﻧﺘﺎج اﻟﻮﺳﻄﺎء اﻟﻤﺆﯾﺪﯾﻦ ﻟﻼﻟﺘﮭﺎﺑﺎت وﻣﻨﮭﻢ 2-COX وiNOS وﻧﺘﺮات اﻷﻧﺴﺠﺔ اﻟﻨﺎﺟﻢ ﻋﻦ اﺳﺘﺨﺪام ﻣﺮﻛﺐ DSS ﺗﻢ ﺣﺠﺒﮫ ﻋﻨﺪ اﺳﺘﺨﺪام ﻧﯿﺮوﻟﯿﺪول.

روّج ﻧﯿﺮوﻟﯿﺪول ﻟﺠﺮﻋﺔ اﻹزاﺣﺔ اﻟﻨﻮوﯾﺔ 2-Nrf ﺑﺎﻻﻋﺘﻤﺎد ﻋﻠﻰ اﻟﺠﺮﻋﺔ. ﻛﻤﺖ زاد ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻣﻦ ﻧﺸﺎط إﻧﺰﯾﻢ ﻣﻀﺎدات اﻷﻛﺴﺪة (SOD وCAT) وﻣﺴﺘﻮﯾﺎت 1-HO وSOD-3و mRNA. أدى ﻋﻼج ﻧﯿﺮوﻟﯿﺪول ﻓﻲ ﺧﻼﯾﺎ 29-HT اﻟﺘﻲ ﺗﻮاﺟﮫ TNF-α إﻟﻰ اﻧﺨﻔﺎض ﻛﺒﯿﺮ ﻓﻲ ﻣﺴﺘﻮﯾﺎت اﻟﻤﻮاد اﻟﻤﺆﯾﺪة ﻟﻼﻟﺘﮭﺎﺑﺎت (1-CXCL و8-IL و CCL2 و 2-COX) ﻋﻠﻰ ﻣﺴﺘﻮى ﺣﻤﺾ اﻟﺮﯾﺒﻮﻧﻮﻛﻠﯿﯿﻚ.

ﻛﯿﻨﺎزات اﻟﺒﺮوﺗﯿﻦ اﻟﻤﻨﺸﻂ ﺑﺎﻟﻤﯿﺘﻮﺟﯿﻦ (MAPKs) واﻟﻌﺎﻣﻞ اﻟﻨﻮوي ﻛﺎﺑﺎ ب (NF-κB) ھﻲ ﻣﺴﺎرات إﺷﺎرات ﻣﮭﻤﺔ ﺗﺸﺎرك ﻓﻲ اﻻﻟﺘﮭﺎب. ﻟﺬﻟﻚ، ﻗﻤﻨﺎ ﺑﺎﻟﺘﺤﻘﯿﻖ ﻣﻦ ﺗﺄﺛﯿﺮ ﻧﯿﺮوﻟﯿﺪول ﻋﻠﻰ آﻟﯿﺔ إﺷﺎرات MAPK وNF-κB. ﺗﺸﯿﺮ ﻧﺘﺎﺋﺠﻨﺎ إﻟﻰ أن ﻓﺴﻔﺮة MAPK (p38 وJNK وERK1/2) و NF-κBﻗﺪ زادت ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻓﻲ ﻧﻤﻮذج اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن اﻟﻨﺎﺟﻢ ﻋﻦ اﺳﺘﺨﺪام DSS

وﺣﻔﺰ LPS ﻓﻲ ﺧﻼﯾﺎ اﻟﺒﻼﻋﺎت ﻣﻦ ﻧﻮع RAW. أدى اﺳﺘﺨﺪام ﻧﯿﺮوﻟﯿﺪول ﻛﻌﻼج ﻓﻲ ﻧﻤﻮذج اﻻﻟﺘﮭﺎب ھﺬا إﻟﻰ ﺗﺜﺒﯿﻂ اﻟﻔﺴﻔﺮة ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻟﺒﺮوﺗﯿﻨﺎت إﺷﺎرات MAPK و NF-κBﻓﻲ ﻛﻞ ﻣﻦ ﻧﻤﻮذج اﻟﻔﺌﺮان وﻓﻲﻏﻼﯾﺎ اﻟﺒﻼﻋﺎت ﻣﻦ ﻧﻮع RAW اﻟﻤﺤﻔﺰة بـ LPS. ﺗﺸﯿﺮ ھﺬه اﻟﻨﺘﺎﺋﺞ إﻟﻰ أن ﻋﻤﻞ ﻧﯿﺮوﻟﯿﺪول اﻟﻤﻀﺎد ﻟﻼﻟﺘﮭﺎﺑﺎت ﯾﺘﻢ ﺑﻮﺳﺎطﺔ ﺗﺜﺒﯿﻂ آﻟﯿﺔ إﺷﺎرات NF-κB / MAPK.

ﯾﺆدي ﺿﻌﻒ اﻟﺤﺎﺟﺰ اﻟﻈﮭﺎري اﻟﻤﻌﻮي إﻟﻰ ﺗﻌﺰﯾﺰ ﻧﻔﺎذﯾﺔ اﻷﻣﻌﺎء ﻓﻲ ﻣﺮض اﻟﺘﮭﺎب اﻷﻣﻌﺎء. ﻟﺬﻟﻚ، ﻗﻤﻨﺎ ﺑﺎﻟﺘﺤﻘﯿﻖ ﻓﻲ ﺗﺄﺛﯿﺮ ﻧﯿﺮوﻟﯿﺪول ﻋﻠﻰ ﺗﺮاﺑﻂ اﻟﺨﻼﯾﺎ اﻟﺨﻠﻮي ﺑﺎﻷﻣﻌﺎء ﻓﻲ اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﺑﺎﺳﺘﺨﺪام ﻧﻤﻮذج اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن اﻟﻨﺎﺟﻢ ﻋﻦ اﺳﺘﺨﺪام ﻣﺎدة DSS (ﻓﻲ ﻧﻤﻮذج اﻟﺠﺴﻢ اﻟﺤﻲ) وLPS اﻟﺬي ﯾﺤﻔﺰ ﻧﻤﻮذج اﻟﻤﺨﺘﺒﺮ Caco-2 monolayers. ﻗﻠﻞ ﻧﯿﺮوﻟﯿﺪول ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻣﻦ ﺗﺮﻛﯿﺰ FITC-dextran ﻓﻲ ﻣﺼﻞ اﻟﻔﺌﺮان ﻣﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﺘﻲ ﺗﻢ اﺳﺘﺨﺪام DSS ﻓﯿﮭﺎ ﻓﻘﻂ ﻣﻤﺎ ﯾﺸﯿﺮ إﻟﻰ ﺗﻘﻠﯿﻞ ﻧﻔﺎذﯾﺔ اﻟﻘﻮﻟﻮن. أظﮭﺮت ﻧﺘﺎﺋﺠﻨﺎ أﯾﻀًﺎ أن LPS ﺗﺴﺒﺐ ﻓﻲ ﺣﺪوث اﻧﺨﻔﺎض ﻓﻲ اﻟﻤﻘﺎوﻣﺔ اﻟﻜﮭﺮﺑﺎﺋﯿﺔ ﺑﻄﺮﯾﻖ اﻟﻈﮭﺎرة (TEER) في Caco-2 monolayers واﻟﺬي ﺗﻢ ﻣﻨﻌﮫ ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻣﻦ ﺧﻼل اﻟﻌﻼج ﺑﺎﺳﺘﺨﺪام ﻧﯿﺮوﻟﯿﺪول. ﻟﻮﺣﻆ أن ﻧﯿﺮوﻟﯿﺪول ﯾﺤﻤﻲ ﺳﻼﻣﺔ اﻟﺤﺎﺟﺰ اﻟﻤﻌﻮي ﻣﻦ ﺧﻼل ﺗﻌﺰﯾﺰ اﻟﺘﻌﺒﯿﺮ ﻋﻦ ﺑﺮوﺗﯿﻨﺎت اﻟﻮﺻﻠﺔ اﻟﻀﯿﻘﺔ ﻣﺜﻞ ﻛﻠﻮدﯾﻦ 1 ، 3 ، 7 ، وإﻛﻠﻮدﯾﻦ ﻓﻲ اﻟﻨﻤﺎذج اﻟﻤﺨﺘﺒﺮﯾﺔ واﻟﺤﯿﻮ ﯾﺔ ﻻﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن. ﺗﻮﺿﺢ ﻧﺘﺎﺋﺠﻨﺎ ﺑﺸﻜﻞ ﺟﻤﺎﻋﻲ أن ﻣﻜﻤﻼت ﻧﯿﺮوﻟﯿﺪول ﺗﺨﻔﻒ اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﻣﻦ ﺧﻼل ﻧﺸﺎطﮭﺎ اﻟﻔﻌﺎل ﻛﻤﻀﺎد ﻟﻸﻛﺴﺪة وﻣﻀﺎد ﻟﻼﻟﺘﮭﺎﺑﺎت ﻓﻲ ﻛﻞ ﻣﻦ ﻧﻤﺎذج اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن ﻓﻲ اﻟﺠﺴﻢ اﻟﺤﻲ وﻓﻲ اﻟﻤﺨﺘﺒﺮ. ﻋﺰز ﻧﯿﺮوﻟﯿﺪول أﯾﻀًﺎ ﺳﻼﻣﺔ ﺗﻘﺎطﻊ اﻷﻣﻌﺎء اﻟﻀﯿﻘﺔ ﻟﻤﻨﻊ اﺧﺘﻼل وظﯿﻔ ﺔ اﻟﺤﺎﺟﺰ اﻟﻤﻠﺤﻮظ ﻓﻲ اﻟﺘﮭﺎب اﻟﻘﻮﻟﻮن.

Download

Share

COinS