| Cold is one of the most important stressors inhibiting the development of livestock in the north.When cold stimulation occurs,animal growth and development are slowed and feed utilization and production performance are also slowed.In addition to methods such as increasing cold protection facilities,improving the heat production capacity of the animal body is also an option.Numerous studies have shown that substances extracted from plants can regulate the development of brown and beige fat and promote thermogenesis,especially in the promotion of non-warmblood thermogenesis.ligustrazine is an extract of the Chinese herb Chuanxiong,which has anti-inflammatory and anti-oxidative stress properties and alleviates liver steatosis and diabetes and its complications.However,ligustrazine has the defect of short half-life,while ligustrazine hydrochloride has higher solubility and stability and longer half-life compared to ligustrazine.Therefore,in this study,we chose to add ligustrazine hydrochloride to investigate its effect on the thermogenic capacity of cold-stimulated mice,and to provide new ideas for animals to cope with cold resistance in northern winter.To investigate the effect of certain concentrations of ligustrazine hydrochloride on the thermogenic capacity of Brown Adipose Tissue(BAT),Inguinal White Adipose Tissue(iWAT)and liver tissues in mice,the experiment will be divided into two phases.In the first phase,we will investigate whether the addition of ligustrazine hydrochloride can promote thermogenesis in BAT and iWAT at a safe concentration range.4-week-old SPF-grade male C57BL/6J mice were randomly divided into four groups,named normothermic group,cold-stimulated group,coldstimulated 50 mg/kg·d ligustrazine hydrochloride group and cold-stimulated 100 mg/kg·d ligustrazine hydrochloride group,respectively.BAT and iWAT and liver tissues were collected from mice after 4 weeks of gavage treatment.The changes in adipocyte size and the presence of inflammatory infiltration and other damages in the liver tissues of BAT and iWAT were observed by HE staining,and tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were detected in the liver tissues of mice by RT-qPCR.The results showed that HE staining indicated that the adipocytes in BAT and iWAT of cold-stimulated mice became smaller after the addition of ligustrazine hydrochloride,and the liver showed no obvious signs of damage.RT-qPCR results showed that the expression of pro-inflammatory cytokines was significantly upregulated in the liver of mice after cold stimulation,and the expression of pro-inflammatory cytokines was suppressed in the liver after the addition of ligustrazine hydrochloride.After determining the effect of ligustrazine hydrochloride addition,50 mg/kg·d was selected as the addition concentration in the second stage experiment.The expression levels of triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and free fatty acid(FFA)in serum were determined by glycerol phosphate oxidase method and double reagent direct method.The changes of BAT,iWAT and liver tissues were observed by HE staining.The changes in the number of liver lipid droplets in cold-stimulated mice after the addition of ligustrazine hydrochloride were observed by oil red O staining.The expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),peroxisome proliferator-activated receptor-α(PPAR-α),peroxisome proliferator-activated receptor-γ(PPAR-γ)and uncoupling protein 1(UCP1)in BAT and iWAT were analyzed using immunohistochemistry,and liver tissues for PGC-1α and UCP1 expression levels in liver tissues.The expression levels of PR Domain-containing 16(Prdm16),Pgc-1α,Ppar-α,Ppar-γ and UCP1 in BAT,and Prdm16,Pgc-1α,Ppar-α,Ppar-γ,Ucp1,transmembrane protein 26(Tmem26)and iodothyronine deiodinase 2(Dio2)in iWAT were measured by RT-qPCR.The protein expression levels of BAT,iWA and liver in thermogenesis and white fat browning were measured by Western Blot.The results showed that the serum levels of TG,TC,LDL-C,and FFA decreased and the HDL-C levels increased in the cold-stimulated mice after the addition of ligustrazine hydrochloride,and the adipocytes of BAT and iWAT in the C50 mg/kg·d group were smaller and the mice did not show inflammatory infiltration and other damage to the liver.The results of iWAT also showed a significant increase in the expression of mRNA and protein related to white fat browning.Immunohistochemical results showed a significant increase in the expression of thermogenesis-related genes in the liver after the addition of ligustrazine hydrochloride.Also,the expression levels of fatty acid oxidation and thermogenesis-related proteins were significantly increased in the liver.These results indicate that the addition of appropriate concentrations of ligustrazine hydrochloride can promote lipid utilization by the body in mice,and this concentration of ligustrazine hydrochloride did not cause significant liver injury and some reduction of lipid content in the liver,and inhibited the expression of pro-inflammatory cytokines in the liver that were elevated by cold stimulation.The expression levels of BAT,iWAT,and thermogenesis-related factors and proteins in the liver of mice were increased. |
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