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Abstract [Objectives]This study was conducted to elucidate the mechanism of action of total glucosides of paeony （TGP） against the liver injury induced by isoniazid （INH） and rifampicin （RFP）， and to provide experimental evidence for rational use of antituberculosis drugs.
[Methods]Liver injury in mice was induced by the combination of INH and RFP in mice. After the mice were given different doses of Total Glucosides of White Paeony Capsules （TGP） for 10 d， the hepatosomatic index， biochemical indices in serum and liver homogenate were measured， and histopathological changes in liver tissue were observed. Glucurolactone was used as the positive control， and 0.9% sodium chloride was used as the negative control in the experiment.
[Results]TGP reduced the activities of alanine transaminase （ALT） and aspartate transferase （AST） in serum and the level of malondialdehyde （MDA） in liver tissue， and increased the level of glutathione （GSH） and the activity of superoxidase dismutase （SOD） in liver tissue.
[Conclusions]TAP has a protective effect against the liver injury induced by INH and RFP.
Key words Total glucosides of paeony； Liver injury； Isoniazid； Rifampicin
Due to the widespread use of drugs， druginduced liver injury （DILI） has become a common and serious drugrelated disease around the world and attracted increasing attention in clinical practice in recent years. Antituberculosis drugs are one of the most important causes of DILI. A systematic review in China showed that[1]three main types of drugsantituberculosis drugs （31%）， alternative drugs （herbal medicines） （19%） and antibiotics （10%） are responsible for DILI in China. The number of tuberculosis patients accounts for 11% of the total population in China， and antituberculosis drug induced injury （ATLI） has become an urgent problem.
Both isoniazid （INH） and rifampicin （RFP） are firstline antituberculosis drugs. According to WHOrecommended standard treatment regimens for tuberculosis， the two drugs are still irreplaceable. They have significant inhibitory effects on both dormant and replicating Mycobacterium tuberculosis， and the administration of the two drugs together has a synergistic effect， which enhances their ability to kill M. tuberculosis， but also increases their hepatotoxicity and even causes severe liver dysfunction. Studies[2]have shown that hepatotoxicity occurs in only 1.6% patients given INH alone， in only 1.1% patients given RFP alone， and in 35% patients given both INH and RFP. The mechanisms of liver injury induced by antituberculosis drugs is quite complex. Administration of hepatoprotective drugs in conventional antituberculosis therapy may reduce liver injury and improve patient compliance. So it is important to screen some drugs that can eliminate hepatotoxicity induced by the combination of INH and RRP， without affecting their antituberculosis activity. White peony （Paeonia lactiflora） is a Chinese traditional medicine. Total glucosides of white paeony （TGP）， including paeoniflorin， hydroxylpaeoniflorin， paeonin， albiflorin， ester glycosides， benzoylpaeoniflorin and other physiologically active components， has various pharmacological effects such as inhibiting autoimmune reactions， antiinflammatory， analgesic and hepatoprotective effects， and is used for treatments of rheumatic and immunological diseases[3]. TGP has protective effects on immunemediated liver injury[4]and CCl4induced acute liver injury[5]in mice， but its effect on liver injury induced by antituberculosis drugs has not been reported.
In this study， the mice liver injury induced by the combination INH and RFP were given the Total Glucosides of White Peony Capsules （Lansen Pharmaceutical Holdings Limited） at different doses for 10 d， before the hepatosomatic index， biochemical indices in serum and liver homogenate were measured， and histopathological changes in liver tissue were observed， to clarify the mechanism of action of TGP against liver injury， and to provide experimental evidence for rational use of antituberculosis drugs.
Materials and Methods
Materials
Animals
Sixty healthy Kunming mice， weighing （25±5） g each， were provided by the Experimental Animal Center of Jiaxing University.
Drugs and reagents
Total Glucosides of White Paeony Capsules （Batch No.： 151032） was produced by Lansen Pharmaceutical Holdings Limited. Isoniazid Tablets （Batch No.： 1510006） and Rifampicin Capsules （Batch No.： 161013） were produced by Xinchang Pharmaceutical Factory， Zhejiang Medicine Co.， Ltd. Glucurolactone Tablets （Batch No.： 201508242） was a product of Jiangsu Tasly Diyi Pharmaceutical Co.， Ltd. The ALT Assay Kit （Batch No.： 20170621）， AST/GOT Assay kit （Batch No.： 20170526）， malondialdehyde （MDA） Assay Kit （Batch No.： 20170616）， glutathione peroxidase （GSHPx） Assay Kit （Batch No.： 20170619）， Coomassie Brilliant Blue （Batch No.： 20170610） and SOD Assay Kit （Batch No.： 20170620） were purchased from Nanjing Jiancheng Bioengineering Institute.
Instruments and equipment
The instruments used in our experiment mainly included AL204 analytical balance[MettlerToledo Instruments （Shanghai） Co.， Ltd]， PALCAXXXLSM2 water purification system （Pall Corporation）， TDL5A Low Speed Centrifuge， JY92IID ultrasonic cell crusher （Shanghai Bilon Instrument Co.， Ltd.）， i3 UVVIS spectrophotometer （Jinan Haineng Instrument Co.， Ltd.） and DHW4 desktop vortex mixer （Taicang Experimental Equipment Factory）. Induction of liver injury and administration of TGP
The sixty Kunming mice were randomly assigned into six groups of 10 animals each. The mice in one group were given 0.9% sodium chloride injection at a dose of 0.5 ml/d during the experiment period （blank control）. The other five groups of mice were given INH （100 mg/kg） and RFP （100 mg/kg） intragastrically at a dose of 0.2 ml/g， to induce liver injury. Two hours later， each of the five groups was given 0.9% sodium chloride injection at a dose of 0.5 ml/d （negative control）， 240 mg/（kg·d） of TGP （highTGP treatment ）， 120 mg/（kg·d） TGP （mediumTGP treatment）， 60 mg/（kg·d） （lowTGP treatment） or 240 mg/（kg·d） glucurolactone （positive control） for 10 d.
Calculation of hepatosomatic index （HSI） and observation of histopathological changes
The entire liver was carefully removed from the body of each mouse and weighed. Hepatosomatic index （HSI） was calculated using the formula： HIS= Liver weight/Body weight （mg/g）. Then， part of liver lobe was sampled， fixed with 10% formaldehyde solution for 24 h， embedded in paraffin， sectioned， stained with HE before being observed under a light microscope for histopathological changes.
Determination of biochemical indices in serum
Blood was sampled from the venous plexus in the orbit of an eye， and the activities of alanine transferase （ALT） and aspartate transferase （AST） in serum were measured using ALT and AST assay kits， following manufacturer’s instruction.
Determination of biochemical indices in liver homogenate
Liver tissue （0.1 g） was sampled， homogenized by adding 0.9 ml saline and centrifuged at 2 500 × g for 15 min （4 ℃） to collect the supernatant. Then， 0.1 ml of the supernatant was taken to measure the activity of superoxidase dismutase （SOD）， and the contents of malondialdehyde （MDA） and glutathione （GSH）， following the manufacturer’s instruction.
Data analysis
All Data were presented as means±standard error， and analyzed with ttest.
Results and Analysis
Effect of TGP on hepatosomatic index
Compared with healthy mice （blank control）， the mice with liver injury had an enlarged liver， as their liver weight and hepatosomatic index were increased to varying degree （P<0.05）.
Effect of TGP on serum AST and ALT activities
Compared with the blank control， the ALT and AST levels of the negative control were significantly increased （P<0.01）， indicating that liver injury was successfully induced by the combination of INH and RFP. Compared with the negative control， glucurolactone treatment and all the TGP treatments significantly inhibited the activities of ALT and AST （P<0.05）， as shown in Table 2.
Effect of TGP on GSH and MDA contents and SOD activity in liver homogenate
As can be seen in Table 3， compared with the blank control， the GSH content and SOD activity of the negative control were significantly decreased， and the MDA content was significantly increased （P<0.01）， suggesting that the animal model of liver injury was successfully established. Compared with the negative control， TGP treatments significantly increased the GSH content and SOD activity， and significantly reduced the MDA content in liver tissue （P<0.05）， and the effect was dosedependent. The results indicated that TGP has a protective effect against the liver injury induced by antituberculosis drugs.
Pathological changes in liver tissue
Fig. 1 shows the histopathological changes of liver tissue of mice in different treatments. In healthy livers （Fig. 1A）， the hepatic cords were regularly arranged； there were no infiltration of inflammatory cells and no hyperplasia of fibrous connective tissue in the hepatic lobule and portal triad. The injured livers showed obvious pathological changes， such as irregularly arranged structure， fuzzy boundaries between hepatic lobules， increased cytoplasmic volume， diffusely distributed necrotic cells and inflammatory cell infiltration （Fig. 1B）. In the liver tissues of glucuronolactonetreated mice （Fig. 1C）， hepatic lobules and portal triads had normal structure， and injured area and inflammatory cell infiltration were both reduced， compared with the negative control. The area and extent of liver injury and inflammatory cell infiltration in both highTGP and mediumTGP treatments were all significantly reduced compared with the negative control （Fig. 1 D and E）， while those of lowTGP treatment were slightly reduced （Fig. 1F）.
（A） Normal liver tissue of healthy mice， （B） liver tissue of negative control， （C） liver tissue of glucurolactone treated mice， （D） liver tissue of highdose TGP treated mice， （E） liver tissue of mediumdose TGP treated mice， （F） liver tissue of lowdose TGP treated mice.
Fig. 1 Histopathological changes of liver tissue of mice in different treatments
Agricultural Biotechnology2018
Discussion and Conclusions
ALT is mainly found in liver cells. Normally， as long as a small amount of ALT is released into the blood， the activity of the enzyme in the serum can be significantly increased. A large quantity of ALT will be released into the blood in response to liver injury. So， it is an important indicator for the diagnosis of liver injury. In the present study， liver injury was induced by the combination of INH and RFP， and as a result， serum ALT level was significantly increased， and subsequent TGP treatments significantly decreased serum ALT level. AST is a protein made by liver cells. Serum AST level is generally low in healthy animals. When liver cells are damaged， they release AST into the blood， resulting in an increase in serum AST level. Our results showed that serum AST level in mice with liver injury was significantly increased， and the level was then greatly decreased by TGP treatments.
GSH， an important antioxidant in animals， has a wide range of physiological functions， can activate a variety of enzymes， and is involved in the TCA cycle and glucose metabolism. It can increase the content of NO in plasma and improve hepatic microcirculation. GSH can inhibit the production of free radicals by providing a reducing agent for glutathione peroxidase and thus protects the liver. Our data showed that GSH level was dramatically decreased in injured liver， and subsequent administration of TGP significantly improved GSH in liver homogenate.
SOD， a free radical scavenger， is widely found in various tissues of organisms and can scavenge superoxide （O2-）， which is cytotoxic， and can cause lipid peroxidation and damage cell membranes. Our data showed that liver injury resulted a decrease in SOD activity in liver homogenate， and the administration of TGP significantly increased SOD level.
MDA results from lipid peroxidation of polyunsaturated fatty acids. And its level can indirectly reflect the extent of cell damage. Our data showed that liver injury resulted an increase in MDA level in liver homogenate， and the administration of TGP significantly decreased the level.
In summary， TGP has a significant therapeutic effect on druginduced liver injury.
References
[1]ZHOU Y. Epidemiology of druginduced liver injury in Chinese population： systematic analysis of 21 789 patients[D]. Chongqing： Army Medical University， 2013.
[2]DONG YZ， HUANG JC， LIN X， et al. Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicininduced liver injury in mice[J]. Elsevier Journal， 2014， 152（1）： 201-206.
[3]LI NQ. A review on pharmacological effects and modern research of Baishao[J]. Clinical Journal of Chinese Medicine， 2017， 20（9）： 137-138.
[4]LIU F. Protective effect of total glucosides of paeony on mice with acute chemical liver injury[J]. Pharmacology and Clinics of Chinese Materia Medica， 2015， 31（4）： 100-102.
[5]XIE YJ， LIU W， ZHANG ZM， et al. Protective effect and mechanism of action of olive leaf extract against Dgalactosamine/lipopolysaccharideinduced acute liver injury[J]. Science and Technology of Food Industry， 2018， 39（3）： 315-319.
Editor： Qingqing YIN Proofreader： Xinxiu ZHU
[Methods]Liver injury in mice was induced by the combination of INH and RFP in mice. After the mice were given different doses of Total Glucosides of White Paeony Capsules （TGP） for 10 d， the hepatosomatic index， biochemical indices in serum and liver homogenate were measured， and histopathological changes in liver tissue were observed. Glucurolactone was used as the positive control， and 0.9% sodium chloride was used as the negative control in the experiment.
[Results]TGP reduced the activities of alanine transaminase （ALT） and aspartate transferase （AST） in serum and the level of malondialdehyde （MDA） in liver tissue， and increased the level of glutathione （GSH） and the activity of superoxidase dismutase （SOD） in liver tissue.
[Conclusions]TAP has a protective effect against the liver injury induced by INH and RFP.
Key words Total glucosides of paeony； Liver injury； Isoniazid； Rifampicin
Due to the widespread use of drugs， druginduced liver injury （DILI） has become a common and serious drugrelated disease around the world and attracted increasing attention in clinical practice in recent years. Antituberculosis drugs are one of the most important causes of DILI. A systematic review in China showed that[1]three main types of drugsantituberculosis drugs （31%）， alternative drugs （herbal medicines） （19%） and antibiotics （10%） are responsible for DILI in China. The number of tuberculosis patients accounts for 11% of the total population in China， and antituberculosis drug induced injury （ATLI） has become an urgent problem.
Both isoniazid （INH） and rifampicin （RFP） are firstline antituberculosis drugs. According to WHOrecommended standard treatment regimens for tuberculosis， the two drugs are still irreplaceable. They have significant inhibitory effects on both dormant and replicating Mycobacterium tuberculosis， and the administration of the two drugs together has a synergistic effect， which enhances their ability to kill M. tuberculosis， but also increases their hepatotoxicity and even causes severe liver dysfunction. Studies[2]have shown that hepatotoxicity occurs in only 1.6% patients given INH alone， in only 1.1% patients given RFP alone， and in 35% patients given both INH and RFP. The mechanisms of liver injury induced by antituberculosis drugs is quite complex. Administration of hepatoprotective drugs in conventional antituberculosis therapy may reduce liver injury and improve patient compliance. So it is important to screen some drugs that can eliminate hepatotoxicity induced by the combination of INH and RRP， without affecting their antituberculosis activity. White peony （Paeonia lactiflora） is a Chinese traditional medicine. Total glucosides of white paeony （TGP）， including paeoniflorin， hydroxylpaeoniflorin， paeonin， albiflorin， ester glycosides， benzoylpaeoniflorin and other physiologically active components， has various pharmacological effects such as inhibiting autoimmune reactions， antiinflammatory， analgesic and hepatoprotective effects， and is used for treatments of rheumatic and immunological diseases[3]. TGP has protective effects on immunemediated liver injury[4]and CCl4induced acute liver injury[5]in mice， but its effect on liver injury induced by antituberculosis drugs has not been reported.
In this study， the mice liver injury induced by the combination INH and RFP were given the Total Glucosides of White Peony Capsules （Lansen Pharmaceutical Holdings Limited） at different doses for 10 d， before the hepatosomatic index， biochemical indices in serum and liver homogenate were measured， and histopathological changes in liver tissue were observed， to clarify the mechanism of action of TGP against liver injury， and to provide experimental evidence for rational use of antituberculosis drugs.
Materials and Methods
Materials
Animals
Sixty healthy Kunming mice， weighing （25±5） g each， were provided by the Experimental Animal Center of Jiaxing University.
Drugs and reagents
Total Glucosides of White Paeony Capsules （Batch No.： 151032） was produced by Lansen Pharmaceutical Holdings Limited. Isoniazid Tablets （Batch No.： 1510006） and Rifampicin Capsules （Batch No.： 161013） were produced by Xinchang Pharmaceutical Factory， Zhejiang Medicine Co.， Ltd. Glucurolactone Tablets （Batch No.： 201508242） was a product of Jiangsu Tasly Diyi Pharmaceutical Co.， Ltd. The ALT Assay Kit （Batch No.： 20170621）， AST/GOT Assay kit （Batch No.： 20170526）， malondialdehyde （MDA） Assay Kit （Batch No.： 20170616）， glutathione peroxidase （GSHPx） Assay Kit （Batch No.： 20170619）， Coomassie Brilliant Blue （Batch No.： 20170610） and SOD Assay Kit （Batch No.： 20170620） were purchased from Nanjing Jiancheng Bioengineering Institute.
Instruments and equipment
The instruments used in our experiment mainly included AL204 analytical balance[MettlerToledo Instruments （Shanghai） Co.， Ltd]， PALCAXXXLSM2 water purification system （Pall Corporation）， TDL5A Low Speed Centrifuge， JY92IID ultrasonic cell crusher （Shanghai Bilon Instrument Co.， Ltd.）， i3 UVVIS spectrophotometer （Jinan Haineng Instrument Co.， Ltd.） and DHW4 desktop vortex mixer （Taicang Experimental Equipment Factory）. Induction of liver injury and administration of TGP
The sixty Kunming mice were randomly assigned into six groups of 10 animals each. The mice in one group were given 0.9% sodium chloride injection at a dose of 0.5 ml/d during the experiment period （blank control）. The other five groups of mice were given INH （100 mg/kg） and RFP （100 mg/kg） intragastrically at a dose of 0.2 ml/g， to induce liver injury. Two hours later， each of the five groups was given 0.9% sodium chloride injection at a dose of 0.5 ml/d （negative control）， 240 mg/（kg·d） of TGP （highTGP treatment ）， 120 mg/（kg·d） TGP （mediumTGP treatment）， 60 mg/（kg·d） （lowTGP treatment） or 240 mg/（kg·d） glucurolactone （positive control） for 10 d.
Calculation of hepatosomatic index （HSI） and observation of histopathological changes
The entire liver was carefully removed from the body of each mouse and weighed. Hepatosomatic index （HSI） was calculated using the formula： HIS= Liver weight/Body weight （mg/g）. Then， part of liver lobe was sampled， fixed with 10% formaldehyde solution for 24 h， embedded in paraffin， sectioned， stained with HE before being observed under a light microscope for histopathological changes.
Determination of biochemical indices in serum
Blood was sampled from the venous plexus in the orbit of an eye， and the activities of alanine transferase （ALT） and aspartate transferase （AST） in serum were measured using ALT and AST assay kits， following manufacturer’s instruction.
Determination of biochemical indices in liver homogenate
Liver tissue （0.1 g） was sampled， homogenized by adding 0.9 ml saline and centrifuged at 2 500 × g for 15 min （4 ℃） to collect the supernatant. Then， 0.1 ml of the supernatant was taken to measure the activity of superoxidase dismutase （SOD）， and the contents of malondialdehyde （MDA） and glutathione （GSH）， following the manufacturer’s instruction.
Data analysis
All Data were presented as means±standard error， and analyzed with ttest.
Results and Analysis
Effect of TGP on hepatosomatic index
Compared with healthy mice （blank control）， the mice with liver injury had an enlarged liver， as their liver weight and hepatosomatic index were increased to varying degree （P<0.05）.
Effect of TGP on serum AST and ALT activities
Compared with the blank control， the ALT and AST levels of the negative control were significantly increased （P<0.01）， indicating that liver injury was successfully induced by the combination of INH and RFP. Compared with the negative control， glucurolactone treatment and all the TGP treatments significantly inhibited the activities of ALT and AST （P<0.05）， as shown in Table 2.
Effect of TGP on GSH and MDA contents and SOD activity in liver homogenate
As can be seen in Table 3， compared with the blank control， the GSH content and SOD activity of the negative control were significantly decreased， and the MDA content was significantly increased （P<0.01）， suggesting that the animal model of liver injury was successfully established. Compared with the negative control， TGP treatments significantly increased the GSH content and SOD activity， and significantly reduced the MDA content in liver tissue （P<0.05）， and the effect was dosedependent. The results indicated that TGP has a protective effect against the liver injury induced by antituberculosis drugs.
Pathological changes in liver tissue
Fig. 1 shows the histopathological changes of liver tissue of mice in different treatments. In healthy livers （Fig. 1A）， the hepatic cords were regularly arranged； there were no infiltration of inflammatory cells and no hyperplasia of fibrous connective tissue in the hepatic lobule and portal triad. The injured livers showed obvious pathological changes， such as irregularly arranged structure， fuzzy boundaries between hepatic lobules， increased cytoplasmic volume， diffusely distributed necrotic cells and inflammatory cell infiltration （Fig. 1B）. In the liver tissues of glucuronolactonetreated mice （Fig. 1C）， hepatic lobules and portal triads had normal structure， and injured area and inflammatory cell infiltration were both reduced， compared with the negative control. The area and extent of liver injury and inflammatory cell infiltration in both highTGP and mediumTGP treatments were all significantly reduced compared with the negative control （Fig. 1 D and E）， while those of lowTGP treatment were slightly reduced （Fig. 1F）.
（A） Normal liver tissue of healthy mice， （B） liver tissue of negative control， （C） liver tissue of glucurolactone treated mice， （D） liver tissue of highdose TGP treated mice， （E） liver tissue of mediumdose TGP treated mice， （F） liver tissue of lowdose TGP treated mice.
Fig. 1 Histopathological changes of liver tissue of mice in different treatments
Agricultural Biotechnology2018
Discussion and Conclusions
ALT is mainly found in liver cells. Normally， as long as a small amount of ALT is released into the blood， the activity of the enzyme in the serum can be significantly increased. A large quantity of ALT will be released into the blood in response to liver injury. So， it is an important indicator for the diagnosis of liver injury. In the present study， liver injury was induced by the combination of INH and RFP， and as a result， serum ALT level was significantly increased， and subsequent TGP treatments significantly decreased serum ALT level. AST is a protein made by liver cells. Serum AST level is generally low in healthy animals. When liver cells are damaged， they release AST into the blood， resulting in an increase in serum AST level. Our results showed that serum AST level in mice with liver injury was significantly increased， and the level was then greatly decreased by TGP treatments.
GSH， an important antioxidant in animals， has a wide range of physiological functions， can activate a variety of enzymes， and is involved in the TCA cycle and glucose metabolism. It can increase the content of NO in plasma and improve hepatic microcirculation. GSH can inhibit the production of free radicals by providing a reducing agent for glutathione peroxidase and thus protects the liver. Our data showed that GSH level was dramatically decreased in injured liver， and subsequent administration of TGP significantly improved GSH in liver homogenate.
SOD， a free radical scavenger， is widely found in various tissues of organisms and can scavenge superoxide （O2-）， which is cytotoxic， and can cause lipid peroxidation and damage cell membranes. Our data showed that liver injury resulted a decrease in SOD activity in liver homogenate， and the administration of TGP significantly increased SOD level.
MDA results from lipid peroxidation of polyunsaturated fatty acids. And its level can indirectly reflect the extent of cell damage. Our data showed that liver injury resulted an increase in MDA level in liver homogenate， and the administration of TGP significantly decreased the level.
In summary， TGP has a significant therapeutic effect on druginduced liver injury.
References
[1]ZHOU Y. Epidemiology of druginduced liver injury in Chinese population： systematic analysis of 21 789 patients[D]. Chongqing： Army Medical University， 2013.
[2]DONG YZ， HUANG JC， LIN X， et al. Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicininduced liver injury in mice[J]. Elsevier Journal， 2014， 152（1）： 201-206.
[3]LI NQ. A review on pharmacological effects and modern research of Baishao[J]. Clinical Journal of Chinese Medicine， 2017， 20（9）： 137-138.
[4]LIU F. Protective effect of total glucosides of paeony on mice with acute chemical liver injury[J]. Pharmacology and Clinics of Chinese Materia Medica， 2015， 31（4）： 100-102.
[5]XIE YJ， LIU W， ZHANG ZM， et al. Protective effect and mechanism of action of olive leaf extract against Dgalactosamine/lipopolysaccharideinduced acute liver injury[J]. Science and Technology of Food Industry， 2018， 39（3）： 315-319.
Editor： Qingqing YIN Proofreader： Xinxiu ZHU