Journal of Acupuncture and Meridian Studies
Volume 4, Issue 4 , Pages 214-219, December 2011

Effects of Manual Acupuncture at GB34 on Carbon tetrachloride-induced Acute Liver Injury in Rats

  • Nobuyuki Tanahashi

      Affiliations

    • Institute of Traditional Chinese Medicine, Suzuka University of Medical Science, 1001-1, Kishioka, Suzuka, Mie 510-0293, Japan
    • ,
  • Juri Shikami

      Affiliations

    • Graduate School of Health Science, Suzuka University of Medical Science, 1001-1, Kishioka, Suzuka, Mie 510-0293, Japan
    • ,
  • Misao Yoneda

      Affiliations

    • Department of Pathology, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu, Mie 514-8507, Japan
    • ,
  • Torao Ishida

      Affiliations

    • Institute of Traditional Chinese Medicine, Suzuka University of Medical Science, 1001-1, Kishioka, Suzuka, Mie 510-0293, Japan
    • Graduate School of Health Science, Suzuka University of Medical Science, 1001-1, Kishioka, Suzuka, Mie 510-0293, Japan
    • Corresponding Author InformationCorresponding author. Graduate School of Health Science, Suzuka University of Medical Science, 1001-1, Kishioka, Suzuka, Mie 510-0293, Japan.

Received 28 May 2011; received in revised form 24 July 2011; accepted 29 August 2011. published online 14 November 2011.

Article Outline

Abstract 

Manual acupuncture at Yanglingquan (GB34) is reported to have a beneficial effect on chronic liver damage. We, therefore, studied the effect of manual acupuncture at GB34 on acute liver damage. Rats were administered carbon tetrachloride (CCl4) or olive oil, and direct manual acupuncture was subsequently performed at GB34 or at a sham point (a nonacupoint). In rats administered with CCl4, the serum levels of aspartate aminotransferase and alanine aminotransferase, the total cholesterol concentration, and the levels of hepatic thiobarbituric-acid-reactive substances were suppressed by acupuncture at GB34 when compared with acupuncture at the sham point. By contrast, there was little histological difference in the liver between acupuncture at GB34 and that at the sham point in rats administered with CCl4. These results suggest that manual acupuncture at GB34 tends to improve acute liver damage but is not sufficient by itself to completely resolve the hepatic injury.

Keywords: acupuncture, carbon tetrachloride, CCl4, GB34, transaminases, Yanglingquan

 

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1. Introduction 

Oriental medicine has a far longer history than modern medicine, and studies of the medical mechanisms of acupuncture are one important aspect providing information on this type of treatment. Acupuncture treatment is reported to be effective not only for pain of the locomotor system but also for various other symptoms, such as those relating to disorders of the digestive, respiratory, and circulatory systems [1]. The mechanisms of this effect are thought to include endogenous pain relief actions, such as a spinal segmental mechanism, a motion control mechanism, somatic reflexes, autonomic reflexes, and neurological and endocrine mechanisms. In particular, an endogenous opioid hormone is released in the central nervous system during acupuncture stimulation [2]. This controls pain by blocking the neural pathway through which the brain recognizes pain. Hence, the effectiveness of acupuncture as an analgesic method seems to be related to blockades of both pain recognition in the brain and transmission of nociception in the spinal cord. Moreover, stimulation of peripheral nerves by acupuncture facilitates blood flow, thereby hastening the removal of substances causing pain. Acupuncture has also been recently reported to be associated with temporary increases in cytokine levels, which stimulate the immune system [3].

The administration of carbon tetrachloride (CCl4) or galactosamine is a proven method of creating an animal model of liver dysfunction. Within 24 hours of administration, CCl4 causes acute liver damage characterized by hepatocellular necrosis in the vicinity of the central vein, infiltration of white blood cells, fat deposition, and remarkable increases in serum transaminase levels. These findings are almost completely restored to a normal state within 72 hours [4].

Acupuncture is effective against various symptoms, and acupuncture at Qimen, Ganshu and Yanglingquan (GB34) is said to be effective for liver dysfunction. Although the literature contains a report on the effect of acupuncture at GB34 for chronic liver damage [5], no studies seem to have been conducted on the effect of acupuncture at this point for acute liver damage. However, acupuncture at Zusanli and Taichong performed following the administration of CCl4 has been reported to be effective against acute liver damage [6]. We, therefore, analyzed the effect of manual acupuncture at GB34 by using a rat model of acute liver damage.

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2. Materials and methods 

2.1. Laboratory animals 

We purchased male Sprague Dawley rats at 8 weeks of age (200–250g) from Clea Japan Inc. (Shizuoka, Japan). We maintained the animals at a room temperature of 22±3°C and a humidity of 60% in a 12-hour light/dark cycle for a 2-week adjustment period after purchase. Rats were given free access to animal feed (CE-2 from Clea Japan Inc.) and water. The approval of the Suzuka University of Medical Science Animal Experiment Ethical Review Board was obtained for the experiments.

2.2. Experimental method 

Laboratory animals were randomly divided into six groups: Normal, CCl4 (–) and no acupuncture; Control, CCl4 (+) and no acupuncture; Sham/CCl4 (–), CCl4 (–) and acupuncture at a sham point selected on the gluteal region, Fig. 1; Sham/CCl4 (+), CCl4 (+) and acupuncture at a sham point; GB34/CCl4 (–), CCl4 (–) and acupuncture at left GB34; and GB34/CCl4 (+), CCl4 (+) and acupuncture at left GB34. Each group consisted of five rats.

Liver injury was induced by intraperitoneal injection of 500mL /L solution of CCl4 in olive oil (2mL/kg) in the CCl4 (+) groups or injection of olive oil in the CCl4 (–) groups. After administration of CCl4 or olive oil, we fixed each rat to a brace and applied manual acupuncture to GB34 or to the sham point three times every 4 hours for 12 hours. This method was followed because the effect of the acupuncture continues for at least 3 hours [7] and because acute liver damage induced by CCl4 shows an invasion of white blood cells, fatty deposition, and an increase in serum transaminase 12 hours after CCl4 administration [8].

Sterilized disposable manual acupuncture needles (0.25mm×30mm, Carbo deluxe, Global Medical Supplies Ltd, Hong Kong) were inserted perpendicularly to a depth of 2–3mm at left GB34 or at a sham point. After the needles had been inserted, they were rotated 90° clockwise and then thrust downward nine times. Nine rotations and nine thrusts constituted one manipulation, and three manipulation units constituted one treatment session. The full manipulation took about 30 seconds. We performed an operation under 10% Nembutal anesthesia 24 hours after CCl4 administration and surgically obtained blood and liver specimens.

2.3. Biochemical analysis 

Blood was centrifuged at 3000 revolutions/minute for 20 minutes to separate the serum. The levels of serum transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were measured as an index of liver damage by using 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride colorimetry (520nm) after conjugation with glutamate dehydrogenase [9], and the serum total cholesterol was measured using a “Cholesterol E-test Wako” kit (Wako Pure Chemical Industries, Osaka, Japan).

Liver specimens were homogenized in 9 volumes of the homogenate buffer (50mM Tris-HCl [pH 7.5], 1.14% KCl, 1mM EDTA) in a Potter-Elvehjem homogenizer from As One Corp. (Osaka, Japan), and this homogenate was used as a crude extract. As an indicator of peroxidative disorder of the liver tissue, we measured the level of crude extract thiobarbituric-acid-reactive substance (TBARS) by using the thiobarbituric acid method [10] and expressed the level as the malondialdehyde quantity.

2.4. Histological analysis 

Liver tissues were fixed in 10% neutral formalin, and the specimens were processed for paraffin embedding after decalcification. Tissue sections were obtained and stained with hematoxylin and eosin.

2.5. Statistical analysis 

The experimental results were expressed as means±standard errors. The behavioral data were analyzed using a one-way analysis of variance (ANOVA) followed by a posthoc Tukey test to compare groups.

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3. Results 

AST and ALT levels in the Sham/CCl4 (–) and GB34/CCl4 (–) groups were low and similar to those in the Normal group. These activities increased significantly in the Sham/CCl4 (+) group when compared with the Normal and Sham/CCl4 (–) groups as shown in Fig. 2 (p<0.05). These results suggest that the liver damage was induced by intraperitoneal injection of CCl4 and not by acupuncture at the sham point or at left GB34. By contrast, the AST and the ALT levels in the GB34/CCl4 (+) group showed only a nonsignificant tendency to increase when compared with those in the GB34/CCl4 (–) group but decreased significantly compared with those in the Control and the Sham/CCl4 (+) groups as shown in Fig. 2 (p<0.05). These results suggest that acute liver damage secondary to CCl4 was limited by performing manual acupuncture at GB34.

  • View full-size image.
  • Figure 2 

    Effect of manual acupuncture at GB34 and at a sham point on serum AST and ALT activities in rats with acute hepatic damage induced by CCl4. * p<0.01 versus Normal CCl4 (–); ** p<0.05 versus Control CCl4 (+). ALT=alanine aminotransferase; AST=aspartate aminotransferase; CCl4=carbon tetrachloride; GB34=Yanglingquan.

The serum total cholesterol levels in the Sham/CCl4 (–) and the GB34/CCl4 (–) groups were high and were similar to those in the Normal group. The serum total cholesterol levels in the Sham/CCl4 (+) group decreased significantly as compared with those in the Normal and the Sham/CCl4 (–) groups as shown in Fig. 3 (p<0.05). These results suggest that hepatic cholesterol synthesis was not affected by acupuncture at the sham point or at left GB34 but was affected by CCl4 injection. By contrast, the serum total cholesterol level in the GB34/CCl4 (+) group showed only a nonsignificant tendency to decrease as compared with that in the GB34/ CCl4 (–) group.

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  • Figure 3 

    Effect of manual acupuncture at GB34 and at a sham point on the serum total cholesterol level in rats with acute hepatic damage induced by CCl4. * p<0.05 versus Normal CCl4 (–). CCl4=carbon tetrachloride; GB34=Yanglingquan.

Disorders of lipid metabolism within the liver have been attributed to injection of CCl4, and in this setting lipid hyperoxidation is accelerated in the hepatic tissue [4]. Therefore, we measured the TBARS levels in the liver tissue as a measure of peroxidative disorder. TBARS levels in the Sham/CCl4 (–) and the GB34/CCl4 (–) groups were low and were similar to those in the Normal group. The TBARS levels in the Sham/CCl4 (+) group were significantly increased as compared with those in the Normal and the Sham/CCl4 (–) groups as shown in Fig. 4 (p<0.05). This suggested that the peroxidative status was not affected by acupuncture at the sham point or at left GB34 but was affected by CCl4 injection. By contrast, the TBARS level in the GB34/CCl4 (+) group showed only a nonsignificant tendency to increase as compared with that in the GB34/CCl4 (–) group, and the recovery trend was more obvious in the GB34/CCl4 (+) group than in the Control and the Sham /CCl4 (+) groups. Therefore, manual acupuncture at GB34 seemed to restrain oxidative stress moderately but not to a statistically significant degree.

  • View full-size image.
  • Figure 4 

    Effect of manual acupuncture at GB34 and at a sham point on liver TBARS concentration in rats with acute hepatic damage induced by CCl4. * p<0.01 versus Normal CCl4 (–). CCl4=carbon tetrachloride; GB34=Yanglingquan; TBARS=thiobarbituric-acid-reactive substance; MDA=malondialdehyde.

Fig. 5 shows the results of hepatic histology. The liver was histologically normal in the Sham/CCl4 (–) and the GB34/CCl4 (–) groups. Compared with the Sham/CCl4 (+) group, the GB34/CCl4 (+) group showed the following tendencies: a reduction in the infiltration of white blood cells, an accumulation of lipids due to cell necrosis and disordered lipid metabolism, and inflammation.

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4. Discussion 

In the present study, we performed manual acupuncture at GB34 because this point is reported to have an effect on various symptoms and conditions, including liver dysfunction. We first administered CCl4 to create an animal model of liver damage, and we confirmed the model by showing the severe features associated with acute hepatitis. Administration of this compound is known to cause hepatocellular necrosis and apoptosis, progressing to hepatic fibrosis or cirrhosis with repeated doses 11, 12, 13. Furthermore, the hepatic Kupffer cells and stellate cells that are damaged by CCl4 have been shown to produce cytokines, such as TNF-α, TGF-β, IL -1, IL-6 or IL-10 14, 15. When manual acupuncture was performed at Zusanli and Taichong after administering CCl4 to rats to induce acute liver damage, reductions in the elevations of the serum AST, ALT, and alkaline phosphatase levels were observed [6]. Furthermore, Yim et al. [5] reported a similar effect on the serum AST, ALT, and alkaline phosphatase activities after performing manual acupuncture at GB34 in a rat model of chronic liver damage. In the present study, we biochemically and histologically analyzed the effect of acupuncture at GB34 on the onset of liver damage that had been induced by using CCl4.

Hepatocellular necrosis can be evaluated by the levels of AST and ALT. Cholesterol is another index of the hepatic function because it is synthesized by the liver. We found that the serum AST and ALT levels increased and that the serum cholesterol level decreased in acute liver damage induced by using CCl4. When CCl4 administration was followed by manual acupuncture at GB34, the serum AST and ALT levels decreased, and the serum cholesterol level tended to increase.

This study suggests that manual acupuncture at GB34 tends to control, or specifically to inhibit, the development of acute liver damage. However, manual acupuncture alone cannot completely inhibit this damage.

The mechanism of action underlying this effect of acupuncture on liver damage remains unknown. The CCl4 used in this study stimulated hepatic Kupffer cells and stellate cells to produce cytokines such as TNF-α, TGF-β, IL-1, IL-6, and IL-10, which induce liver damage [14].

In a general clinical setting, manual acupuncture treatment is effective not only in palliation of pain but also in reinforcing natural healing mechanisms, including the immune response, thereby improving overall health status. In particular, manual acupuncture stimulation has been reported to have the potential to change the production of cytokines, such as IL-10 or IFN-γ, by the immune system: a mechanism that seems to be mediated through the autonomic nervous system and endogenous opioid production 2, 16, 17, 18. For instance, after acupuncture, TNF-α and IL-10 are increased, and IL-2 and IFN-γ are reduced in CCl4-damaged liver tissue 19, 20, 21.

The protective effect against liver damage of manual acupuncture stimulation at GB34 is, therefore, thought to be associated with an interaction between the nervous and the immune systems. However, the precise action mechanism, including the immune components, by which acute liver damage is mitigated needs to be clarified.

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PII: S2005-2901(11)00027-6

doi:10.1016/j.jams.2011.09.012

Journal of Acupuncture and Meridian Studies
Volume 4, Issue 4 , Pages 214-219, December 2011

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