Effects Of Manganese Deficiency On Chondrocytes Development-related Factors In Broiler Tibia Growth Plate

To investigate the effects of manganese deficiency on chondrocytes development andsearch for the signaling mechanism of tibia disorder development caused by manganesedeficiency, chondrocytes proliferation and apoptosis and cartilage extracellular matrixdegradation in tibia growth plate were tested. Chondrocytes development-related factors weremeasured and analyzed, in order to provide the foundation for pathogenesis of perosis causedby manganese deficiency.Ninety1-day-old Arbor Acres chicks were randomly divided into three groups and fedon control diet (60mg Mn/kg diet) and manganese deficiency diets (40mg Mn/kg diet,manganese deficiency group I;8.7mg Mn/kg diet, manganese deficiency group II),respectively, with a24-h light, and were given free access to food and water. All chicks werekilled by cervical dislocation in compliance with institutional guidelines and in a humanefashion at the age of42days. The growth plates were removed. After measuring the thicknessof growth plate and width of proliferative zone and hypertrophic zone of growth plate, theywere dissected out and immediately fixed in4%formaldehyde, embedded in paraffin andsectioned at5μm. Paraffin sections were mounted on poly-l-lysine coated glass slides andwere used for HE staining and immunohistochemistry. Approximately0.2g of growth platepieces from each sample were immediately frozen in liquid nitrogen for RNA extraction.Some pieces were immediately fixed in2.5%glutaral for24h, and then embedded in epoxyresins. Sections were cut and viewed under transmission electron microscope. Chondrocyteapoptosis was estimated by TUNEL staining. Gene expression of P21and Bcl-2, andexpression of P21, Bcl-2, cbfα1, CDMP-1, MMP-1, Cx43and PTHrP proteins were analyzedby quantitative real time RT-PCR and immunohistochemistry, respectively. The results areshown as follows:(1) Compared with control group, manganese deficiency groups showed significantly lowerin proliferative zone and hypertrophic zone width (P<0.05), and also showed significantlylower in growth plate width.(2) There is a remarkable increase (P<0.05) in the expression of P21, Cx43and MMP-1and a remarkable decrease (P<0.05) in the expression of cbfα1and PTHrP in manganesedeficiency groups. Expression of CDMP-1decreased, but there was no significant difference (P>0.05).(3) Chondrocyte mitochondrial outer membrane and mitochondrion cristae were damagedcompared with normal mitochondria. TUNEL staining showed that there is a significantincrease in the apoptotic chondrocytes rate in manganese deficiency groups (P<0.05).Compared with the control group, the Bcl-2relative expression levels of the manganesedeficiency groups decreased. Though there is no significant difference in the expression ofBcl-2between manganese deficiency groups (P>0.05), there is a significant differencebetween control group and manganese deficiency groups (P<0.05).The results indicate that manganese deficiency induced the collagenous fiber of cartilageextracellular matrix degradation. Meanwhile, manganese deficiency inhibited chondrocytesproliferation and promoted chondrocytes apoptosis. It is speculated that chondrocyteapoptosis induced by manganese deficiency may be dependent on mitochondria-mediatedapoptosis. The signaling way of disorder development of growth plate caused by manganesedeficiency may be by Ihh/PTHrP pathway.