Salvia Miltiorrhiza Reinstates Growth Plate Width, Reduces Liver Oxidative Stress and Toxicity in Avian Tibial Dyschondroplasia

One hundred and fifty 1-day-old Arbor Acres broilers (AAC) (46.5 ±0.5 g) were purchased from commercial hatchery (Chia Tai Animal Husbandry Co. Ltd., Wuhan, China) and raised under standard hygienic conditions that included cleaning, disinfecting, free access to clean drinking water and standard feed, constant lighting, optimum humidity and temperature in proper well-ventilated room. The chickens were divided into 3 equal groups, Group 1 (n=50) received control feed and groups 2 and 3 (n=50 each) were fed thiram from day 3 (Shanghai Macklin Biochemical Co. Ltd., China) @ 50 mg/kg of feed to induce TD until end of the experiment day-15. After induction of TD, the group 3 birds received treatment with SM (trade name Danshen) injections prepared from the dried root extract of SM was purchased from Wuhan Dinghui Chemical Co., Ltd. SM was dissolved in dimethyl sulfoxide (DMSO) and diluted to make stock solution. SM (Danshen) group were given i.p. injection @ 10 mg/kg/day of SM through intra-peritoneal route from day 8 until end of the experiment on day 15. All the experiments were conducted after the approval of institutional Animal Welfare and Care, Ethics Committee guideline of Huazhong Agricultural University, Wuhan, China.

Blood samples (n=10 each) were collected by cardiac puncture on day 8 and 15 from each group. Blood samples were centrifuged at 3000×g for 20 min to separate serum which was stored at -70°C, until subsequent use to determine the serum ALT, AST level and ALP activities. All chicks were kept for 15 days then slaughtered by cervical dislocation (Nabi et al., 2016a) to collect liver samples from each group. The liver samples were stored at -70°C for subsequent analysis of SOD, GSH-Px activity and MDA content. Briefly, whole liver tissues samples were chopped into small pieces on ice, a homogenate was prepared in 10mM phosphate buffered saline (10 ml PBS/0.1 g of tissue) and centrifuged at 3500 rpm/min for 10 min at 4°C. The supernatant was collected and stored for determination of the SOD, GSH-Px activities and MDA content by commercial reagent kits (Nanjing Institute of Biological Engineering Inc., Jiangsu, China) following manufacturer’s instructions. The SOD and GSH-Px results

presented in Unit per milligram of protein (U/mg protein), while MDA contents were expressed in nanomoles/ gram wet weight of liver tissue (nmoles/g). The values of AST, ALT and ALP activities were presented in unit per liter (U/L). All methods were performed according to our previous studies (Li et al., 2007; Shahzad et al., 2014; Nabi et al., 2016, b).

Data were analyzed with one way ANOVA by student t-test to compare the differences between mean values of control and treatment groups. The differences were considered statistically significant if P < 0.05. All statistical analyses were performed using SPSS 19.0 software and results are presented as means±SEM.

The morphological examination revealed the reduced growth rate and lameness in thiram-induced TD group as compared to control. Birds regained their growth rate, ability to stand and walk after the treatment with SM (Fig. 1A). Tibial growth plate morphology indicated that width of the proximal tibial growth plates was markedly enlarged in the TD group as compared to control. However, administration of SM medicine resulted in significant decrease in TD and growth-plate width (Fig. 1B).

A significant (P<0.05) decrease in serum ALP activity along with an increase in serum ALT and AST contents were observed in thiram-fed birds as compared to control group. However, SM administration caused a significant increase in ALP level (P<0.05) with a decrease in serum ALT and AST levels significantly (P<0.05) as compared with thiram only fed group. The antioxidants SOD and GSH-Px were significantly decreased (P<0.05), while the MDA contents was increased in TD-affected chickens as compared to control group. However, the intra peritoneal SM administration restored and increased antioxidant markers (SOD and GSH-Px) with a decrease in MDA contents significantly (P<0.05) (Fig. 2).

SM is used as an anti-osteoporotic drug in traditional Chinese medicine. It has been widely used for the microcirculatory disturbance and for the treatment of cardiovascular and cerebrovascular diseases, liver dysfunction, renal deficiency and vascular complications of diabetes (Han et al., 2008). Traditional Chinese Medicine (TCM) approach offers a holistic solution to prevent and eradicate bone disease. SM extract also enhances bone formation through increasing angiogenesis and osteoblastic activity and decreasing osteoclastic activity (Wong and Rabei, 2008). In the current study, treatment with 10 mg/kg of SM significantly attenuated TD, decreased growth plate width and eliminated lameness in broiler chicken.

Some authors described that thiram is an oxidant agent, ultimately increased oxidative stress on liver and lead to decreased in antioxidant (SOD and GSH-Px) activities (Li et al., 2007; Marikovsky, 2002). MDA is well-known cell membrane injury marker, lipid peroxidation resulting in release of MDA product in the end (Marikovsky, 2002;Nabi et al., 2016b; Shahzad et al., 2014). Thiram-induction can lead to TD disease causes liver damages with increasing of liver damages indicators (AST and ALT) in blood and toxicity (Gupta and Amma, 1993; Li et al., 2007; Shahzad et al., 2014; Nabi et al., 2016a).

TD is characterized by lack of calcification and causes failure of epiphyseal growth plate chondrocytes hypertrophy. ALP measurement in serum is primary marker of bone formation and TD is associated with significant decreased ALP level (Genin et al., 2012). SM treatment significantly restored ALP level in blood serum. ALP level is deficient in TD but its level was restored to normal level both in affected area and in blood after the treatment with SM with concurrent resolution of TD (Nabi et al., 2016a; Genin et al., 2012; Shahzad et al., 2014).

Liver toxicity and oxidative stress is considered to mutual pathological mechanism due to several inflammatory or pathological conditions in animals (Li et al., 2015; Marikovsky, 2002). Diverse factors induce toxicity and oxidative stress and the liver is the principal detoxifying organ that directly impairs the production performance of chicken. Administration of natural antioxidants in case of liver damages or oxidative stress significantly a rational strategy to cure oxidative imbalance and antioxidant capabilities.

Salvia miltiorrhiza appears to act as a natural traditional Chinese medicine with oxidant scavenging capabilities. Administration of SM to dyschondroplastic birds abrogates lameness, reduces oxidative stress markers and hepatic injury and appears to have health benefits and therapeutic efficacy against avian TD in broiler chicken.

This study was supported by the National key Research and Development Program of China (Project No 2017YFD0502200) and National Natural Science Foundation of China (No. 31460682).

Authors declare that they have no competing interests.