Therapeutic Effects Of Traditional Chinese Medicines Against Tibial Dyschondroplasia Via PI3K/AKT/HIF-1? Signaling Pathway

Tibial dyschondroplasia is defined as non-vascularize and non-mineralized white opaque mass or plaque in the proximal growth plate of tibia bone.It is a common skeletal abnormality in avian species which happens frequently in rapidly growing broiler chickens.It has been noticed as economically important disease because it is devastating poultry industry by carcass loss at processing plant due to bone fracture.On other hand it is an animal welfare problem because birds cannot walk and move properly due to serious lameness.1.Effects of tetramethylpyrazine on tibial dyschondroplasia by regulating HIF-1? and VEGFTibial dyschondroplasia(TD)is basically a pathological abnormality in which cartilage fails to become bone due to non-vascular growth plate(GP).The proximal growth plate of tibia bone becomes non-vascularize and non-mineralize with white opaque mass.Tetmethylpyramizine(TMP)is mostly used for the treatment of hypertension and neurovascular diseases,but there is no report regarding its protective effect against tibial chondrodysplasia.For this purpose we selected 210 day old chickens and distributed them in three groups equally(n= 70);control group(normal feeding during experiment period),TD group(thiram treatment was given just 3-7 days)and TMP group(thiram treatment from 3-7 days and then TMP treatment from 8-18 days).The chickens(n= 15)were randomly selected and slaughtered at various days(7,10,14 and 18).The chicken mortality,performance indicators parameters,tibia bone parameters,TD score,histology of tibial growth plate,expression of HIF-1alpha and VEGF genes and proteins were studied.Our results demonstrated that in TD group production parameters including weight gain,feed intake and FCR were decreased compared to control group during the study period.Tibial bone parameters including length,width and weight were lowered non-significantly(P>0.05)throughout the experiment period,whereas size of tibial GP was increased significantly(P<0.05)in TD chickens compared to control group on day 7,10 and 14.Furthermore,TD group showed more TD score as compared to control group,while TMP treatment reduced the TD score significantly(P<0.05).The histopathological analysis of tibial growth plate indicated uneven columns of chondrocytes cells surrounded with few numbers of blood vessels in TD group;whereas TMP group demonstrated well conserve chondrocytes columns with plenty of blood vessels.HIF-1alpha and VEGF genes and proteins expressions were significantly(P<0.05)up-regulated in TD affected chickens.But TMP treatment significantly(P<0.05)decreased the expression level of both genes and proteins of HIF-1alpha and VEGF.In general,the results showed that TMP treatment decreased the width of GP,lameness,TD score and expression of HIF-1alpha and VEGF,while increased the growth related parameters.So,our study suggests that TMP play an important role in angiogenesis for the recovery and impairment of GP in TD chickens by regulation of HIF-1alph/VEGF.2.Ligustrazine remodels the toxicity in thiram-induced tibial dyschondroplasia chickens: involvement of new molecules modulating integrin beta-3Thiram(tetramethyl thiuram disulfide)is a dithiocarbamate pesticide,it is commonly used to protect crop against pests and fungi.It is used for the protection of seed during storage and transportation.Over use of thiram as pesticide and environmental contamination of crop with thiram caused tibial dyschondroplasia in chickens.The chicken can get tibial dyschondroplasia on feeding thiram containing diet.The present study conducted to investigate the effects of ligustrazine(tetramethylpyrazine or TMP)to remodel the toxicity in thiram fed TD chickens.A total of 210 chickens were distributed randomly into three equal groups just like above mentioned study.During the experiment,the lameness,severity and incidence of TD,tibia index changes,growth plate pathology,serum parameters,antioxidant imbalance and expression of integrin beta 3(ITGB3)were studied.The results of our study showed that during serum analysis ALP was lowered while the contents of ALT and AST were more in thiram fed TD chickens.Antioxidant parameters analysis indicated that the values of SOD,T-AOC,AND GSH-Px were decreased significantly and contents of MDA were increased significantly in TD group compared to control and TMP groups.However,TMP administrations recovered the lameness and reinstated the serum biochemical parameters and antioxidant imbalance.TMP also diminished the TD lesion and histopathological changes along with the reduction of tibia index,incidence of TD as well as severity of TD in TD afflicted birds.ITGB3 gene and protein expression was significantly decreased in thiram fed chickens compared to control group.The administration of TMP to tibial dyschondroplasia afflicted chickens enhanced the expression significantly compared with TD group on various days.So,TMP is an important agent to treat tibial dyschondroplasia by reducing the damaging effect on chondrocytes and modulating ITGB3 expression.3.Protective effect of astragaloside IV to inhibit tibial dyschondroplasia on vascular endothelial cell based PI3K/Akt/HIF-1? signalingAstragaloside IV(AST-IV)is a natural glycoside,which is extracted from Astragalus membranaceus.It has been widely used as anti-cancer,anti-inflammatory,anti-oxidative,and immune-regulatory effects,and these actions have been reported to be implicated in protecting cells apoptosis.AST-IV promotes osteogenesis but its protective affects about TD has not been study yet.So,we designed this study to use AST-IV for the treatment of TD through PI3K/Akt/HIF-1? pathway.For this purpose a total of 180 Arbor Acres chickens were equally divided into control group,TD group and Astr(AST-IV treated chickens)group.The control group was fed a standard normal diet ad libitum,while the TD group and Astr group were fed a normal diet supplemented with thiram @50 mg/kg of feed from 3 to 7 days;after 7 days,the thiram was removed and AST-IV was administered orally @ 50 mg/kg body weight to Astr group.During the experiment,the mortality rate,production parameters,physiological changes,biochemical criterion,liver antioxidant enzymes,and tibial parameters along with genes and proteins expression related to PI3K/Akt/HIF-1? signaling pathway were examined in all chicken groups.The results showed GP size was increased significantly,while mortality and lameness was prominent in TD chickens but difference was not significant.The level of ALP,SOD,GSH-Px and T-AOC were decreased,while AST,ALT and MDA were increased after the induction of TD.AST-IV treatment significantly increased the level of ALP,SOD,GSHPx and T-AOC,and decreased the AST,ALT and MDA contents.Furthermore,AST-IV administrations restored the growth performance and GP size,whereas decreased the mortality,tibia lesions and tibial cartilage damage in chickens.The gene and protein expression of VEGF,VEGFR1,HIF-1alpha and COX-2 were more in TD chickens compared to control group.After the administration of AST-IV to chickens,the expression of VEGF,HIF-1alpha and COX-2 were decreased.Akt and PI3 K were less expressed in TD group as compared to control group.Whereas,AST-IV treatment increased the expression of these genes near to control group.Altogether,RT-qPCR,western blotting and immunohistochemistry results reveal that AST-IV regulate the expression of various genes and proteins related to PI3K/Akt/HIF-1? signaling pathway.