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    A review of time-effect research on acupuncture in experimental rats/mice in the recent 10 years

    2020-02-14 10:45:38LuHai魯海HuJiahui胡佳慧HanLisha韓李莎ZhangChunhong張春紅WangYu王煜
    關(guān)鍵詞:李莎王煜胡佳

    Lu Hai (魯海), Hu Jia-hui (胡佳慧), Han Li-sha (韓李莎), Zhang Chun-hong (張春紅), Wang Yu (王煜)

    1 First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

    2 Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China

    3 Academic Experience Inheritance Studio of Master of Traditional Chinese Medicine Shi Xue-min, Tianjin 300193, China

    4 National Clinical Medicine Research Center of Traditional Chinese Medicine, Tianjin 300193, China

    Abstract

    Keywords: Acupuncture Therapy; Acupuncture Effect; Acupuncture-moxibustion Mechanism; Needle Retaining; Dose Response Relationship, Acupuncuture-moxibustion; Treatment Course; Rats; Mice

    Rats and mice are small mammalian rodent animals. Due to its small size, fast reproduction, easy breeding, easy access, minor individual differences, similarity to human physiological functions, and large number of inbred lines, mutant lines, and closed groups, it is currently widely used as an experimental animal in medicine, agronomy and related fields[1]. Acupuncture, as an effective external treatment method, is widely favored by scholars in China and abroad[2-3]. A large number of rats/mice experiments have been carried out to study the mechanism of acupuncture efficacy and to screen the best operation parameters. With the advancement of science and technology and the development of modern time acupuncture in recent decades, the ‘time-effect relationship’ of acupuncture has been deeply studied. The ‘time-effect’ refers to the law that the acupuncture effect changes with time and is the main scientific basis for establishing the acupuncture time parameters[4]. Due to the differences in the occurrences, developments, and outcomes of different diseases, the acupuncture time-effect varies from disease to disease, which brings challenges to the basic research of acupuncture time-effect law. It also leads to scattered and isolated studies of acupuncture time-effect experiments, and the experimental parameters are single and discontinuous. Although current research has made some progress, a completed system has not been built up. This article took the acupuncture process as the starting point, summarized the research progress of the time-effect law of acupuncture based on experimental rats/mice in the recent 10 years in order to clarify the current acupuncture foundation, the trend of experimental research on the time-effect relationship, and to provide new ideas for time-effect acupuncture research.

    1 Time Factors Before Acupuncture

    1.1 Intervention timing

    As an interventional method, acupuncture has not reached a consensus on the best intervention timing. However, early acupuncture interventions focusing on multi-target, multi-level and multi-channel interventions within an effective ‘time window’ are crucial to the treatment outcome[5]. The screening study of acupuncture experiments in rats/mice at different intervention timings provides a basis for early diagnosis and the best intervention time for early intervention.

    Zhu SX,et al[6]observed the effect of acupuncture at different timings on the pregnancy outcome of controlled superovulation mice, and the results showed that acupuncture applied on the same day of human chorionic gonadotropin (HCG) injection was the best treatment timing, and can increase the secretion of insulin-like growth factor 1 (IGF-1) in the endometrium, improve the clinical pregnancy rate and the average number of implanted sites. In the study of acupuncture intervention on traumatic brain injury (TBI) model mice, it was also found that the intervention of acupuncture on the first day after the model was made can best upregulate brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), B cell lymphoma-2 (Bcl-2) protein, and corresponding gene mRNA contents in the injured brain tissues[7-8]. She LJ,et al[9]compared the effects of the electroacupuncture (EA) intervention on pain memory model rats at different timings. The results showed that EA can effectively reduce the occurrence of pain memory induced by the second injection of carrageenan, and early treatment had a better effect than that in the late stage. Lu ZX,et al[10]studied multi-time EA for lumbar multifidus muscle injury in rats, and the results showed that the early-stage intervention (1-2 d) of EA at Weizhong (BL 40) can significantly increase insulin-like growth factor 1 receptor (IGF1R), down-regulate the expression of insulin-like growth factor binding protein 3 (IGFBP3), and promote the repair of multifidus muscle. Some studies have found that pre-acupuncture is more effective than immediate acupuncture in regulating beta-endorphin (β-EP) content and heat shock protein 70 (HSP70) expression in the hypothalamus and pituitary in dysmenorrhea rats. The effects in improving renal function and anti-oxidative free radical damage are more significant in simulated weightlessness rats[11-12]. However, the study of EA intervening vascular dementia (VD) mice found that EA can significantly improve VD mice's learning and memory ability, better activate hippocampal superoxide dismutase (SOD), norepinephrine (NE), dopamine (DA), and serotonin 5-hydroxytryptamine (5-HT) contents; reduce malondialdehyde (MDA) and nitric oxide synthase (NOS) contents. The intervention effect on the third day of modeling was better than that on the day of modeling[13-14].

    It can be seen that the effect of intervention varies with the timing of intervention, and the timing effect of acupuncture intervention differs among different disease models. However, in general, early (1-3 d) acupuncture intervention is the key to improving the efficacy. It is positively related to the timing of acupuncture intervention. The earlier the intervention, the better the efficacy. Early acupuncture treatment can be used in the early stage of the disease. Pre-acupuncture is emphasized as it may ‘prevent disease before it happens’ and should be carried out in an ultra-early stage.

    1.2 Acupuncture moment

    The theory of acupuncture timing believes that body function is related to time, the Ying-nutrient and Wei-defensive qi, qi and blood flow in the body and is affected by the yin and yang, day and night, and the four seasons. Therefore, the best curative effect can only be obtained by applying acupuncture at the right time. The rhythm of regulating the body's qi and blood at different times has also been verified in rats.

    Wang YJ,et al[15]compared the effect of acupuncture on the blood pressure in spontaneous hypertension rat (SHR) in Chen period (from 7 a.m. to 9 a.m.), You period (from 5 p.m. to 7 p.m.) and Zi period (from 11 p.m. to 1 a.m.), and the results showed that the reductions in the total systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR were most significant in Chen period (from 7 a.m. to 9 a.m.). Zhao CJ,et al[16]observed the effects of Ling Gui Ba Fa (Midnight-noon Ebb-flow theories) on the serum SOD and MDA in guinea pigs. The results showed that the SOD activity had the largest increase in Mao period (from 5 a.m. to 7 a.m.), and the smallest increase in Wu period (from 11 a.m. to 1 p.m.); the content of MDA had the largest decrease in Wu period (from 11 a.m. to 1 p.m.), and the smallest decrease in Hai period (from 9 p.m. to 11 p.m.). Liu XG,et al[17-18]compared the effects of acupuncture in different periods on the circadian rhythm of the body temperature of rats. The results showed that acupuncture in Mao period (from 5 a.m. to 7 a.m.) delayed the rhythm peak phase by 22.36° and can help most in the recovery process of rhythmic phase of yin deficiency rats. Acupuncture in You period (from 5 p.m. to 7 p.m.) would advance the peak phase by 39.32° and can effectively restore the rhythmic phase of yang deficiency rats as well. Zhou MJ,et al[19]found that acupuncture in Shen period (from 3 p.m. to 5 p.m.) had a better effect than that in Zi period (from 11 p.m. to 1 a.m.) in down-regulating transient receptor potential vanilloid 4 (TRPV4) of transient receptor potential ion channel of inflammatory dorsal root ganglion in rats and the effect was also better in raising the pain threshold. While the effect was inferior on TRPV4 in spinal cord tissue to that of acupuncture in Zi period (from 11 p.m. to 1 a.m.).

    Although the best acupuncture treatment effect can be obtained using the point-open method, the acupuncture time varies depending on the diseases and points. In addition, if the study uses a disease model with circadian rhythm characteristics, the opposite living habits of rodents need to be taken into consideration.

    2 Time Factors During Acupuncture

    2.1 Operation time

    Acupuncture is a scientific medical method operated by practitioners; the appropriate operation time is the key to achieving the optimal amount of stimulation. The operation time of acupuncture usually refers to the duration of applying manipulation after qi arrives at a single point. The operation time of EA usually refers to the duration of stimulating a single point with a certain frequency, waveform, and intensity.

    Li YJ,et al[20]found the optimal time of acupuncture for the pathological morphology of brain tissues in middle cerebral artery occlusion (MCAO) rats with the mind-refreshing and orifice-opening needling method at Shuigou (GV 26) was 5 s (twisting frequency: 3 times/s). Another study optimizing the effect of mind-refreshing and orifice-opening needling method at Neiguan (PC 6) on cerebral blood flow, cerebral infarct volume and neurological deficit in MCAO rats showed the best time parameters were 60 s (EA frequency: 2 Hz)[21]and 180 s (manual operation frequency: 1 time/s)[22]. In addition, Min ZY,et al[23]screened the best operation time parameter for acupuncture at Shenshu (BL 23) to treat rats with kidney-yang deficiency as 60 s (twisting angle: 90°, frequency: 120 times/min). Zhou J,et al[24]studied the optimal combination of operation time and EA parameters for analgesic effect in inflammatory pain rats, frequency/wave width/time: 2 Hz/50 μs/3 s, followed by 100 Hz/200 μs/3 s, the two frequencies switched automatically; intensity/time: 1 mA/15 min and 2 mA /15 min.

    In the acupuncture experiments, the best time and parameters vary greatly due to different diseases, operation methods, and the operators’ habits in manual operation. EA intervention is more objective, but the best time and parameters still vary according to individuals, diseases, weights, current frequencies, and intensities, etc. The general principle for the formation of the optimal amount of stimulation is the higher the frequency of hand operation, the higher the frequency of the EA wave, and the greater the intensity, the shorter the operation time, vice versa.

    2.2Needle-retaining time

    Needle-retaining time refers to the duration from inserting the needle into the point to removing the needle from the point. EA retaining time mainly refers to the duration of electrical stimulation. Needle retaining can wait for the qi arrival, adjust qi, reinforce the deficiency and reduce the excess, and reinforce the healthy qi and eliminate the pathogenic factors. Needle- retaining time is an important parameter in studying the ‘time-effect’ relationship of the optimal induction period of acupuncture, and an important indicator of acupuncture ‘dose’ accumulation. In the current clinical practice, needle retaining is generally 20-30 min.

    Cui JM,et al[25]compared the immediate effects with on the effects of retaining needles for 10 min, 20 min and 30 min on tail-flick pain threshold in mice with acupuncture at Zusanli (ST 36). The results showed that the tail-flick incubation period of the mice with 20-minute needle retaining reached the maximum value, that is, acupuncture achieved the maximum analgesic effect. Therefore, the best effect of manual acupuncture analgesia is produced by 20 min of needle retaining. Xu X,et al[26]compared the effects of EA for 30 s and 3, 5, 10, 15, 30 min on transforming growth factor-α (TGF-α) and HSP70 in rats with an acute gastric mucosal injury. The results showed that the long-term retaining groups (15 min and 30 min) had higher integrated optical density values of TGF-α and HSP70 expressions compared with the other four groups[26], along with reduced gastric mucosal injury index, plasma endothelin (ET), and white blood cells, interleukin (IL)-6, IL-8 and other inflammatory factors[27], and they more effectively repaired acute gastric mucosal injury in rats. However, there was no significant difference between the 15 min and 30 min groups. The ‘research on the effect of different needle-retaining times combinations of EA at Hegu (LI 4) and Sanyinjiao (SP 6) on the uterine contraction and endocrine hormones of rats in late pregnancy’ showed that the best needle-retaining time was 25 min: 20 min at bilateral Hegu (LI 4) followed by 5 min at bilateral Sanyinjiao (SP 6)[28-29].

    The optimal needle-retaining time for acupuncture is still 20-30 min, by which the acupuncture reaches the optimal stimulation amount. With the extension of the needle-retaining time, the acupuncture effect shows a downward trend, which provides an experimental basis for the clinical needle-retaining time. However, whether the effect of acupuncture increases again when the needle stays for more than 60 min still needs further study. Considering the sequence of applying acupuncture at different points as well as the synergistic or antagonistic effects of points compatibility, an independent time-effect relationship study and verification are needed.

    3 Time Factor After Acupuncture

    3.1 Interval

    The post-acupuncture effect after the peak effect of a single acupuncture treatment reflects the decay process of acupuncture effect in the body, similar to the half-life and residual period of drug metabolism in the body and maintaining a certain ‘dose’ of acupuncture effect requires repeated stimulation. Acupuncture interval (acupuncture frequency) is a key indicator demonstrating the effect after acupuncture.

    Zhu ZH,et al[30]compared the effects on total antioxidant capacity (T-AOC), MDA, and SOD of acupuncture at Zusanli (ST 36) at different intervals (twice a day, once a day, once every 2 d, once every 4 d) in mice with spleen deficiency. The results showed that compared with the model group, the T-AOC of the mouse kidney tissues in the once-a-day and twice-a-day groups increased significantly, and the peak value was found in the once-a-day group. The content of MDA in the myocardial tissues of each acupuncture group decreased significantly, and the peak value was in the once-a-day group. The red blood cell SOD activity in the once-a-day group and twice-a-day group both increased significantly, and the peak value was found in the twice-a-day group, but not significantly different from that in the once-a-day group. Therefore, acupuncture at Zusanli (ST 36) once a day had the optimal effect. Wang WJ,et al[31]compared the effects of 3, 6, 12, and 24 h EA intervals on hypothalamic opioid peptide gene expression in rats with inflammatory pain. The results showed that treatment at 24-hour interval can significantly reduce the pain level and increase the pain threshold of rats with inflammatory pain, and promote the expression of pro-opiomelanocortin and pre- enkephalin mRNAs in hypothalamus. EA had an obvious analgesic effect on the inflammatory pain rat model, and repeated treatment at an interval of 24 h (once a day) had the best analgesic effect.

    Based on current experimental research results, in order to consolidate and improve the therapeutic effect of acupuncture, the optimal interval time is 24 h, that is, the best frequency of receiving acupuncture is once a day.

    3.2 The course of acupuncture

    The course of acupuncture is also an important influencing factor for maintaining post-acupuncture effect. Continuous treatment over a period of time can ensure effective control of the disease. However, there is no independent research on the relationship between the acupuncture treatment course and the curative effect. The experimental study has different regulations on the number of treatment days.

    Yang QH,et al[32]compared the effects of different EA treatment courses on the formation of substance P (SP) in the hippocampus of Alzheimer’s disease (AD) mice. The results showed that compared with the group of 2 courses of EA (7 d/course), the SP in the group of 3 courses of EA was significantly reduced. Amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1) expression levels were significantly reduced, and the insulin-degrading enzyme (IDE) expression level was increased. Therefore, EA can reduce the deposition of SP in the hippocampus of AD mice and improve its learning and memory and space exploration ability, and the treatment was preferably 3 courses (3 weeks). Dong WG,et al[33]compared the effects of acupuncture for 1, 2, 3, 4, and 5 courses (14 d/course) on the motor function and astrocyte structure in cerebral infarction rats. The results showed that in the second, third, fourth, and fifth courses, only a few rats recovered more successfully in the acupuncture group than in the model group. Both astrocyte proliferation and neurons had progressive changes at the end of the first, second, and the third courses, and in the fourth and fifth courses tended to stabilize. Therefore, acupuncture for 3 courses (6 weeks) was the best effective in enhancing the activity of astrocytes and promoting the recovery of limb function in paralyzed rats. Similar studies have found that 1, 3, 6, and 9 courses of EA (6 d per course) can promote the functional recovery of paralyzed limbs and the reconstruction of the plasticity of astrocytes and neurons in rats with cerebral infarction. There is a trend that the longer the treatment (6 weeks, 9 weeks), the better the efficacy[34].

    The relationship between intervention time and efficacy hasn’t been proved and the length of treatment is mostly based on experience. The optimal course of acupuncture treatment for AD mice is 3 weeks while the best course of treatment for rats with cerebral infarction is 6 weeks. How to determine the course of acupuncture treatment in different disease models? Is there a linear relationship between the length of treatment and the effect? If so, which type of the linear relationship is it? These questions still need a large number of studies to answer.

    4 Discussion

    Throughout the time-effect study of acupuncture intervention in experimental rats/mice, factors including intervention timing, intervention moment, operation time, needle-retaining duration, interval, and treatment course need to be discussed. However, quantitative research on the time of acupuncture intervention in experimental rats is inconclusive.

    According to the mainstream perspective of the quantitative study we discussed above, the earlier the acupuncture intervention, the better. Different disease models and different points should be treated at different moments in the day. Operation time, regardless of manual operation or EA, the optimal parameters vary greatly according to individual differences, diseases, manipulative methods, the wave frequencies and intensities, etc. However, the experimental rats/mice have a small body size, so the operation time should not be too long, the best needle-retaining time is 20-30 min. Considering the combined effect of different applying sequences, the needle-retaining time should be further determined by orthogonal design for different diseases. The ideal acupuncture frequency is once a day. The intervention courses for different diseases are different, and the linear relationship between the treatment duration of acupuncture intervention for different diseases and the efficacy should be explored. Seeking the best course of treatment can save time and effort while achieving the best amount of stimulation.

    The time-effect experimental study of acupuncture is the only way to standardize modern acupuncture and moxibustion science. The effect in the future study should be based on the law of points and the rules of acupuncture regulation, combined with modern imaging technology, based on the biochemistry, physiology, and data analysis, visualizing parameter data, forming a new visual evidence chain[35], and determining the optimal values of the parameters which impact the effect of acupuncture intervention, and emphasizing the time-effect study that focuses on the whole progress of one single disease (dominant diseases), thus to provide evidence for clinical practice and subject development.

    Conflict of Interest

    The authors declare that there is no conflict of interest.

    Acknowledgments

    This work was supported by National Natural Science Foundation of China (國家自然科學(xué)基金項目, No. 81704148); Scientific Research Project of Tianjin Municipal Education Commission (天津市教育委員會科研計劃項目, No. 2017KJ149).

    Received: 24 October 2019/Accepted: 18 December 2019

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