聶 雨，張斌斌，黃 波，杜玉枝，吳 芹，徐尚福,劉 杰

(1.遵義醫(yī)學(xué)院 基礎(chǔ)藥理教育部重點實驗室和特色民族藥教育部國際合作聯(lián)合實驗室，貴州 遵義 563099;2.中國科學(xué)院西北高原生物研究所 青海省藏藥藥理學(xué)和安全性評價研究重點實驗室，青海 西寧 810001)

基礎(chǔ)醫(yī)學(xué)研究

70味珍珠丸對CCl4致急性肝損傷小鼠的保護作用及機制研究

聶 雨1，張斌斌1，黃 波1，杜玉枝2，吳 芹1，徐尚福1,劉 杰1

(1.遵義醫(yī)學(xué)院 基礎(chǔ)藥理教育部重點實驗室和特色民族藥教育部國際合作聯(lián)合實驗室，貴州 遵義 563099;2.中國科學(xué)院西北高原生物研究所 青海省藏藥藥理學(xué)和安全性評價研究重點實驗室，青海 西寧 810001)

目的70味珍珠丸是珍寶類藏藥，由金、銀、珍珠、珊瑚、麝香、牛黃、藏紅花等 70余味動物、植物和礦物藥組成，主要用于心血管系統(tǒng)疾病，其保肝作用尚未見報道，本實驗研究其對四氯化碳致急性肝損傷小鼠的保護作用及機制研究。方法①將小鼠隨機分為5組，分別為正常對照組、模型對照組、70味珍珠丸(150、500 mg/ kg,po x 7d)和齊墩果酸(25 mg/ kg，ipx3 d)組。于末次給藥6h后腹腔注射0.1% CCl4(10 mL/kg) ，建立急性肝損傷模型。 于CCl4造模18 h后時取材測定小鼠血清 ALT和AST活性，HE 染色觀察肝臟病理組織學(xué)變化，RT-PCR觀察肝毒性及炎性相關(guān)因子表達水平。②隨機取小鼠5只，單獨給藥70味珍珠丸(500 mg/kg,po)，連續(xù)4 d，RT-PCR檢測抗氧化損傷通路基因金屬硫蛋白1(MT-1)、核因子相關(guān)因子2(Nrf2)、NAD(P)H:醌氧化還原酶1(Nqo1)及血紅素氧合酶-1(HO-1)、抗氧化物酶GSH相關(guān)基因(Gclc，GST-mu，GST-pi)的表達水平。結(jié)果與正常對照組比較，模型組CCl4肝損傷小鼠ALT，AST 的活性明顯升高，與模型組比較，70味珍珠丸高劑量組(500 mg/kg)能顯著降低CCl4所引起的ALT，AST 的升高(P<0.05)。70味珍珠丸高劑量組及齊墩果酸組小鼠肝臟病理損傷程度明顯減輕，肝細胞腫脹、變性壞死程度減輕，灶區(qū)壞死明顯減少，炎性細胞浸潤程度有所改善。CCl4肝損傷模型組肝毒性相關(guān)基因Gadd45和Gadd153以及炎性相關(guān)因子MIP2,IL-6表達明顯升高， 70味珍珠丸高劑量組及齊墩果酸組(500 mg/ kg)能明顯減少Gadd45、Gadd153以及炎性因子MIP2,IL-6的表達(P< 0.05)；單獨給藥組與正常組比較，抗氧化損傷通路基因金屬硫蛋白1(MT-1)表達明顯升高(P<0.01)，核因子相關(guān)因子2(Nrf2)、NAD(P)H:醌氧化還原酶1(Nqo1)及血紅素氧合酶-1(HO-1)表達明顯升高(P<0.05)，抗氧化物酶GSH相關(guān)基因(Gclc，GST-mu，GST-pi)表達明顯升高(P<0.05)。結(jié)論70味珍珠丸對CCl4致小鼠急性肝損傷具有一定的保護作用，其保護機制與激活MT -1 、Nrf2、GSH抗氧化損傷的通路有關(guān)。

70味珍珠丸；四氯化碳；氧化損傷；金屬硫蛋白；Nrf2抗氧化通路

70 Wei Zhen-Zhu-Wan (70W) was developed in the middle of the fifteenth century,and continued to use till today.70W is listed in the 2015 edition of Pharmacopoeia of the People's Republic of China[1].70W is a famous Tibetan medicine,composed of herbo-metallic mixtures prepared by special processing methods of Tibetan medicine[1-3].The clinical observations showed many therapeutic effects of 70W,including sedation[4],anti-convulsion,improvement of learning and memory[5].70W is effectively used in the treatment of cardiovascular diseases[6],such as cerebral ischemia[7],cerebral concussion[8],hypertension[9],heart disease[10]and gastrointestinal diseases.However,its hepatoprotective effect has not been reported; this study aimed to examine its protective effects against carbon tetrachloride-induced liver injury in mice.

1 Material

1.1 Reagents 70W was provided by Tibet Tibetan Medicine Manufacture (Lhasa,China).Product with code number approved by SFDA Z54020062 and detail is listed in Pharmacopoeia of China[1].70W was prepared by grinding the pill into powder,adding distilled water to prepare the suspension for oral administration; Carbon tetrachloride (CCl4) was purchased from Sigma-Aldrich (St.Louis,MO).All other chemicals were commercially available reagents.

1.2 Animal Male Kunming mice SPF-grade were purchased from Animal Experimental Center of Third Military Medical University (Chongqing,China,SCXK-2012-0011).Mice were maintained in the Barrier environment facilities (Certificate No.SYXK 2011-004) at Zunyi Medical University,with controlled environment (22±1℃,50±2% humidity and 12 h:12 h light:dark cycle) and free access to purified water and standard laboratory chow.All animal care and experimental protocols are complied with the Animal Management Guidelines of the Chinese Ministry of Health and approve by Animal Use and Care Committee of Zunyi Medical University.

2 Methods

2.1 Mice were randomly divided into 5 groups,respectively as normal control,CCl4model,CCl4+ 70W (150,500 mg/kg,po x 7d) and oleanolic acid (25 mg/kg,ip x 3d as positive control) groups.Twenty-four hr after the last dose of 70W administration,mice were intraperitoneally given 0.1%CCl4(10 mL/kg 18 h),and liver injury was examined by serum enzyme activities and by pathology[11-12].Livers were collected and stored at -80°C for analysis.

2.2 The activity of ALT and AST was determined by kits according to the manufacturer’ protocol (Jiancheng,Nanjing,Lot#20160126).Liver samples were fixed in 10% formalin prior to routine processing and paraffin embedding.Liver sections (3 um) were stained with hematoxylin and eosin and evaluated for histopathology.

2.3 Real-time PCR Approximately 50-100 mg of tissues was homogenized in 1 ml TRIzol (TakaRa Biotechnology,Dalian,China) and total RNA was extracted according to manufacturer’s instructions.The quality and quantity of RNA were determined by the Nano Drop (Thermo Scientific,ND-2000,USA),with 260/280 ratio (>1.8).Total RNA was reverse transcribed with a High Capacity Reverse Transcriptase Kit (Applied Biosystems,Foster City,CA,USA).The primers were designed with Primer3 software and listed (Table 1).The 15 μl PCR reaction mix contained 3 μl of cDNA (10 ng/μl),7.5 μl of iQTM SYBR Green Supermix (Bio-Rad Laboratories,Hercules,CA),0.5 μl of primer mix(10 μM each),and 4 μl of ddH2O.After 5 min denature at 95°C,40 cycles will be performed:annealing and extension at 60°C for 45 seconds and denature at 95°C for 10 seconds.Dissociation curve was performed after finishing 40 cycles to verify the quality of primers and amplification.Relative expression of genes was calculated by the 2-△△Ct-method and normalized to the house keeping gene β-actin or expressed as % of controls[13].

Tab1OligonucleotidesequencesofprimersforRT-PCRanalysis

GeneNumberForwardReverseβ-actinV01217TGACCGAGCGTGGCTACAGGGGCAACATAGCACAGCTTCTGadd45Gadd153MIP2L28177NM_007837NM_009140GCTGCTCAACGTAGACCCCGTCACTACTCTTGACCCTGCGCCTCAACGGAAGAACCAAAGAGCAGCTAGCCGACCCGGGTTGGACTGGAATCTGGAGAGCGACTCAGACAGCGAGGCACATCIL-6J03783GCCCACCAAGAACGATAGTCAGAAGGCAACTGGATGGAAGTCTMT-1Nrf2Nqo1Ho-1GclcGST-muGST-piNM_013602BC026943BC004579M33203BC019374NM_010358D30687CTCCGTAGCTCCAGCTTCACCGAGATATACGCAGGAGAGGTAAGATATCCTTCCGAGTCATCTCTAGCACCTCACTGGCAGGAAATCATCTGGCCACTATCTGCCCAATTCTCCCGACTTTGACAGAAGCTGGGCATCTGAAGCCTTTTGAGGAGCAGCAGCTCTTCTTGGCTCGACAATGTTCTCCAGCTTTCTGCAGCTTCCAGCTTCTTGCCTCGTGGAGACGCTTTACATAGTCTGACACGTAGCCTCGGTAATTGCTCTGGGTGATCTTGTGGATCTGGTCACCCACGATGAA

2.4 Statistical analysis Data were expressed as mean and standard error.The SPSS 16 software was used for statistical analysis.Data were analyzed using a one-way analysis of variance (ANOVA),followed by Duncan’s multiple range test.Pvalue < 0.05 was considered statistically significant.

3 Results

3.1 70W protected against CCl4-induced liver injury CCl4produced liver injury can be evaluated by increased serum enzyme activities and histopathology.Compared with model group,the high dose of 70W and oleanolic acid reduced the serum ALT and AST activities induced by CCl4(P<0.05,Fig 1) .

#Compared with control group P<0.05; *Compared with model group P<0.05.Fig 1 Effect of 70 Wei Zhen-Zhu-Wan (70W) on serum ALT and AST (±s，n=10).

3.2 Liver histopathology CCl4produced liver injury,as evidenced by hepatocellular necrosis,apoptosis,and inflammation (B),70W (500 mg/kg) (D) and oleanolic acid groups (E) reduced these pathology lesions(Fig 2).

A：Control group；B：CCl4 model group；C：CCl4+70 W 150 mg/kg ; D：CCl4+70 W 500 mg/kg); E：CCl4+oleanolic acid 25 mg/kg.Fig 2 Histopathology (HE，×200)

3.3 70W reduced CCl4-induced toxicity gene expression

3.3.1 DNA damage is a hallmark of CCl4-induced liver injury[14-15].The results of RT-PCR showed that in the model group,the expression of liver toxicity-related genes was increased.Compared with the model group,the expression of Gadd45 and Gadd153 in 70W(500 mg/kg) and oleanolic acid group decreased significantly (P<0.05,Fig 3).

#Compared with model group P < 0.05，*Compared with Model group P<0.05.Fig 3 Effect of 70 Wei Zhen-Zhu-Wan (70W) on the expression of toxicity-related genes in mice (±s，n=10)

3.3.2 CCl4-induced liver injury is characterized by inflammation and pro-inflammatory gene expression The results of RT-PCR showed that in the model group,the expression of liver inflammatory cytokines increased.Compared with the model group,the expression of MIP2 and IL-6 in 70W(500 mg· kg-1) and oleanolic acid groups decreased significantly (P<0.05,Fig 4).

3.4 70W alone increased hepatic antioxidant components The hepatic antioxidant genes are mainly metallothionein (MT) and Nrf2 pathway genes[19-20].Compared with the control group,70W alone increased MT(3x) and activated Nrf2 (12%),Nqo1 (60%),Ho-1 (60%),Gclc (80%),GST-mu (40%),GST-pi (40%).The expressions of all these genes were significantly increased (P<0.05,Tab 2).

# Compared with model group P < 0.05，*Compared with Model group P <0.05.Fig 4 The effect of 70 Wei Zhen-Zhu-Wan (70W) on the expression of inflammatory cytokines in mice(±s，n=10)

Tab 2 The effect of 70 Wei Zhen-Zhu-Wan (70W) on the expression of antioxidative genes

genesControl70W(500mg/kg)MT-1163±49.4484±86.9*Nrf286.0±5.38101±3.92*Nqo1Ho-1GclcGST-muGST-pi21.0±2.9083.6±6.1423.2±3.4947.7±3.7668.4±1.2332.9±2.96*133±7.23*41.9±1.90*68.1±7.31*93.1±7.05*

4 Discussion

The present study showed that 70W has protective effects against CCl4hepatotoxicity,as evidenced by decreased ALT,AST and improved histopathology.Oral administration of 70W alone can significantly increase liver antioxidant capacity.This is the first time to provide a pharmacological evidence for the hepatoprotective effect of 70W.

Oxidative stress plays an important role in CCl4hepatotoxicity[16].The trichloromethyl radical can react with oxygen to form the trichloromethyl peroxy radical CCl3OO·,a highly reactive species that initiates the chain reaction of lipid peroxidation,which attacks and destroys polyunsaturated fatty acids,affecting mitochondria,endoplasmic reticulum (ER),and plasma membranes,resulting in the loss of cellular calcium sequestration and homeostasis and increased permeability of cells leading to cell death[19].70W high dose and oleanolic acid significantly reduced liver injury,while 70W low dose group was ineffective,showing that 70W and oleanolic acid can protect against CCl4-induced oxidative liver injury,and this effect is dose-dependent.

CCl4induced liver injury is associated with high expression of inflammatory factors[19].MIP2 is a chemotactic cytokine secreted mainly by macrophages.In the early stage of inflammation,MIP2 was secreted to cause inflammatory cell infiltration,leading to cell damage in the late stage.It was shown that the apoptosis of the cells might be related to the early release of MIP2 in the early stage of inflammation.IL-6 is a kind of hormone-like peptide,which is an important immune regulator[20].IL-6 can act on target cells by remote secretion,and it is more important in inflammatory response.The present results showed that the expression of IL-6 and MIP2 was increased by CCl4.The expression of IL-6 and MIP2 was decreased in the 70W high dose and oleanolic acid group,indicating that 70W and oleanolic acid could reduce the expression of inflammatory factors to reduce liver injury.

The mechanism of protection against liver injury induced by CCl4is related to increased MT-1 and Nrf2[19-20].Induction of MT and Nrf2 is the main mechanism of oleanolic acid hepatoprotection[21].Pretreatment of animals with 70W could induce body defense mechanisms,a phenomenon called “program the liver”[22],which in turn makes animals resistant to toxicant insult.MT-1 is known as one of the most effective free radical scavenger,has strong antioxidant activity,and plays an important role in the process against various liver damage[20].Nrf2 is an important transcription factor related to cell self-protection.Nrf2 is normally inhibited by Keapl,in the case of oxidative stress and Keapl is dissociated,and Nrf2 is moved to nuclear to bind antioxidant response element (ARE) to produce antioxidant gene expression,including the phase II detoxification enzymes.The present results showed that MT and Nrf2 was significantly increased by 70W pretreatment,indicating that 70W could “program the liver” to induce the antioxidant capacity of the tissue to eliminate excessive free radicals.The increased expression of Nrf2 further drives the expression of Nqo1,Gclc,GST-mu,GST-pi and HO-1 to produce protective effect.

In summary,70W has protective effects on acute liver injury produced by CCl4in mice in a dose-dependent manner and reduces the expression of DNA damage inducible genes Gadd45,Gadd153 and inflammatory genes MIP2,IL-6.These effects appear to be mediated via induction of metallothionein and Nrf2 antioxidant genes.

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(編輯：譚秀榮)

Protective effects of 70Wei Zhen-Zhu-Wan against carbon tetrachloride induced liver injury in mice

Nie Yu1，Zhang Binbin1，Huang Bo1，Du Yuzhi2，Wu Qin1，Xu Shangfu1，Liu Jie1

(1.Key Laboratory for Basic Pharmacology of Ministry of Education and the Joint International Research Laboratory of Ethnomedicine of Ministry of Educationof Zunyi Medical University,Zunyi Guizhou 563099,China; 2.Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation,Northwest Plateau Institute of Biology of Chinese Academy of Sciences,Xining Qinghai 810001,China)

Objective70 Wei Zhen-Zhu-Wan (70W) is a famous Tibetan medicine listed in the 2015 edition of Pharmacopoeia.This study examined its protective effects against carbon tetrachloride (CCl4)-induced liver injury in mice.MethodsMice were given 70W (150,500 mg/kg,po x 7d),or oleanolic acid (25 mg/kg,sc x 3d),or distilled water.Six hours after the last dose,mice were intraperitoneally given 0.1% CCl4(10 mL/kg for 18 h),and liver injury was examined.Results70W protected against CCl4induced liver injury,as evidenced by decreased serum enzyme activities and improved histopathology.The high dose of 70W and oleanolic acid reduced the serum ALT and AST levels induced by CCl4,and ameliorated hepatic inflammation and necrosis.CCl4-induced overexpression of DNA-damage inducible genes (Gadd45,Gadd153) and inflammation genes (MIP2,IL-6) were reduced by 70W and oleanolic acid.70W alone activated liver detoxification genes such as metallothionein and the Nrf2 antioxidant pathway genes.Conclusion70W had protective effects against acute liver injury induced by CCl4in mice,and these effects appear to be mediated via induction of metallothionein and Nrf2 antioxidant genes.

70 Wei Zhen-Zhu-Wan; carbon tetrachloride; oxidative damage; metallothionein; Nrf2 antioxidant pathway

青海省重點實驗室發(fā)展專項(NO：2014-Z-Y02)。

劉杰，男，博士，研究員，研究方向： 藥物代謝與毒理，E-mail:jieliu@zmc.edu.cn。

R285.5

A

1000-2715(2017)04-0358-06

[收稿2017-04-03;修回2017-06-27]