Protective Effect of Shentong Zhuyutang-containing Serum on Chondrocytes in Rats with Knee Osteoarthritis Based on AMPK/Sirt1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Shentong Zhuyutang-containing serum against oxidative stress and apoptosis in chondrocytes of rats with knee osteoarthritis （KOA）. MethodFifty male rats were orally administered with normal saline， low-， medium-， and high-dose Shentong Zhuyutang （1.73， 3.46， 6.92 g·kg-1）， and glucosamine sulfate （0.3 g·kg-1） for two weeks. Serum samples were collected after the treatment period. The KOA model was established， and chondrocytes were isolated and randomly divided into normal group， model group， low-， medium-， and high-dose Shentong Zhuyutang-containing serum groups， and glucosamine sulfate group. During the chondrocyte culture， adenosine monophosphate （AMP）-activated protein kinase （AMPK） inhibitor Compound C （10 μmol·L-1） was added， and the cells were divided into normal group， model group， Shentong Zhuyutang-containing serum group， and Compound C + Shentong Zhuyutang-containing serum group. Cell proliferation was detected using 5-ethynyl-2'-deoxyuridine （EdU） staining. Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）. Reactive oxygen species （ROS） levels were measured using DCFH-DA probe. Glutathione （GSH） and malondialdehyde （MDA） levels were determined using the colorimetric method. Real-time polymerase chain reaction （PCR） was used to measure the mRNA expression levels of matrix metalloproteinase-3 （MMP-3）， MMP-13， type Ⅱ collagen （Col Ⅱ）， and Aggrecan. Western blot was performed to measure the protein expression of phosphorylated （p）-AMPK and silent information regulator factor 1 （Sirt1）. ResultCompared with the normal group， the model group showed a significant decrease in chondrocyte proliferation rate， GSH activity， Col Ⅱ and Aggrecan mRNA expression， p-AMPK and Sirt1 protein levels （P<0.01）， and increased ROS levels， MDA content， TUNEL-positive cell rate， and MMP-3 and MMP-13 mRNA expression （P<0.01）. Compared with the model group， Shentong Zhuyutang-containing serum increased the number of EdU-positive cells， GSH activity， Col Ⅱ and Aggrecan mRNA expression， p-AMPK and Sirt1 protein levels in KOA rat chondrocytes （P<0.05， P<0.01）， and decreased the TUNEL-positive cell rate， ROS levels， MDA content， MMP-3 and MMP-13 mRNA expression （P<0.05， P<0.01）. Compared with the Shentong Zhuyutang-containing serum group， the Compound C + Shentong Zhuyutang-containing serum group showed significantly reduced p-AMPK and Sirt1 protein expression， GSH activity， Col Ⅱ and Aggrecan mRNA levels （P<0.01）， and increased TUNEL-positive cell rate， ROS levels， MDA content， MMP-3 and MMP-13 mRNA levels （P<0.01）. ConclusionShentong Zhuyutang-containing serum attenuates oxidative damage and reduces apoptosis in chondrocytes of rats with KOA， and its protective effect may be associated with the activation of the AMPK/Sirt1 signaling pathway.

Analysis on Quality Attribute of Substance Benchmarks of Shentong Zhuyutang Based on Supramolecular "Imprinting Template" Theory / 中国实验方剂学杂志

ObjectiveBased on the supramolecular "imprinting template" theory， the autonomous action law of the component groups of Shentong Zhuyutang in the preparation process of medicinal materials-decoction pieces-formulas was studied to clarify the quantitative transfer law of its quality attributes. MethodUltra performance liquid chromatography（UPLC） fingerprint of Shentong Zhuyutang was established with mobile phase of 0.4% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-2.5 min， 100%A； 2.5-6 min， 100%-96%A； 6-15 min， 96%-92%A； 15-25 min， 92%-88%A； 25-35 min， 88%-75%A； 35-50 min， 75%-65%A； 50-60 min， 65%-50%A； 60-65 min， 50%-30%A； 65-70 min， 100%A） and detection wavelength of 235 nm， and the total statistical moments， information entropy and primary feeding amount of fingerprint of medicinal materials， decoction pieces and benchmark samples were calculated. Dry extract rate of the benchmark samples， the transfer rates and the addition parameters of medicinal materials-decoction pieces-formulas were calculated. ResultSimilarities of the total statistical moments of UPLC fingerprint of 15 batches of medicinal materials and decoction pieces were>0.89， the relative standard deviations（RSDs） of information entropy of UPLC fingerprint of 12 medicinal materials and decoction pieces were<10%. RSDs of total first-order moment（MCRTT） and information entropy of Shentong Zhuyutang（medicinal materials） were 5.5% and 2.3%， while the RSDs of MCRTT and information entropy of Shentong Zhuyutang（decoction pieces） were 4.8% and 2.6%， respectively. The dry extract rate of 45 batches of Shentong Zhuyutang was 17.2%-20.2%. The transfer rate of medicinal materials to decoction pieces was within the range of data fluctuation， which was 70%-130% of the average value. The overall transfer rates of medicinal materials to decoction pieces and decoction pieces to benchmark samples were 101.8% and 83.0%， respectively. ConclusionThe quality properties of Shentong Zhuyutang benchmark samples can be studied by total statistical moment analysis and primary feeding amount analysis， which can confirm the supramolecular "imprinting template" theory to a certain extent.

Herbal Textual Research on Myrrha in Famous Classical Formulas / 中国实验方剂学杂志

In order to provide the basis for the development of famous classical formulas containing Myrrha， the name， origin， quality evaluation， harvest and processing of Myrrha were systematically researched by consulting the ancient herbal and medical books， combining with the modern related literature. According to textual research， the results showed that Commiphora myrrha was the main base in ancient times， which was produced in Somalia， Ethiopia and northern Kenya. In addition， raw and fried products of Myrrha were the commonly used specifications in ancient herbal medicine， which are still used today. Nowadays， Myrrha， fried Myrrha and vinegar-processed Myrrha were the commonly used specifications. Among the three specifications， Myrrha is the raw products after cleaning， fried Myrrha is a kind of processed products， which has relevant records in ancient materia medica and is still used today. Vinegar-processed Myrrha is a new processing specification in modern times. Based on the research results， it is suggested that Myrrha in Shentong Zhuyutang should be the purified raw Myrrha in accordance with the 2020 edition of Chinese Pharmacopoeia.

Textual Research on Ancient and Modern Literature Analysis and Research of Classical Prescriptions for Shentong Zhuyutang / 中国实验方剂学杂志

The Shentong Zhuyutang is recorded in an ancient medical book named Yilin Gaicuo， and it is one of the classical prescriptions. Based on the literature reviewed，it is generally found that the Shentong Zhuyutang is evolved from the prescription named Chentongsan that recorded by Danxi Xinfa. Statistics on the dosage of clinical research on this prescription in recent years， reference to the textbook and pharmacopoeia dose， at the same time to respect the original dosage， verify historical changes， and ensure the safety of prescriptions， at the same time consider the pharmacy's dispenses， the author recommends that the clinical dosage of Shentong Zhuyutang could be 3 gram of Gentianae Macrophyllae Radix， 6 gram of Chuanxiong Rhizoma， 10 gram of Persicae Semen， 10 gram of Carthami Flos， 6 gram of Glycyrrhizae Radix Et Rhizoma， 3 gram of Notopterygii Rhizoma Et Radix， 5 gram of Myrrha， 12 gram of Angelicae Sinensis Radix， 5 gram of Cyperi Rhizoma，12 gram of Cyathulae Radix， and 6 gram of Pheretima used as a reference. The efficacy of the compound preparation can be marked as promoting blood circulation， removing blood stasis， removing wind and dampness， and relieving pain. At present， most of its research is focused on clinical research and experimental research， and there are few literature on the research of Shentong Zhuyutang from the level of key information verification. The diseases it treats include more than 10， such as Gubi， Pibi， Xuebi， shechuanchuan， headache， postpartum pain and dysmenorrhea， especially in the study of Gubi， which means that it has a huge market demand and broad development prospects in orthopedic diseases， the core point of "blood stasis and cold dampness evil" should be closely held in the course of treatment. Some scholars deem that Shentong Zhuyutang should not be used the method of Force Sweat in the treatment of Bizheng，while others believe that this prescription should be used with caution in menstruation， pregnancy and blood deficiency. Modern pharmacological studies have shown that Shentong Zhuyutang has obvious anti-inflammatory， relieve pain， anticoagulan， nerve and bone protection. In view of the fact that there are less researches on Shentong Zhuyutang except for Gubi， this suggests that the mechanism of this prescription treat other diseases has a relatively broad research space.

Clinical Efficacy of Modified Shentong Zhuyutang in Treatment of Acute Discogenic Low Back Pain Due to Wind-Cold and Stasis Obstruction Syndrome / 中国实验方剂学杂志

Objective:To evaluate of modified Shentong Zhuyutang combined with celecoxib capsules in the treatment of acute discogenic low back pain due to wind-cold and stasis obstruction sydrome. Method:One hundred and forty eligible patients were randomly divided into control group （70 cases） and observation group （70 cases）. Patients in both groups took the celecoxib capsules orally， 200 mg/time. On this basis， patients in the control group were further treated with Tongluo Huoxue capsule orally， 6 capsules/time， 3 times/day， while those in the observation group received the modified Shentong Zhuyutang， 1 bag/day， for three weeks. The short-form McGillpain questionnaire （SF-MPQ）， including pain rating index （PRI）， present pain intensity （PPI）， and visual analog scale （VAS）， Oswestry disability index （ODI）， wind-cold obstruction and stasis syndrome， and abilities of daily living （ADL） scoring and the Schober's test were conducted before and after treatment. The tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， IL-6， thromboxane B₂ （TXB₂）， and 6-keto prostaglandin F₁<italic>_α</italic> （6-keto-PGF₁<italic>_α</italic>） levels before and after treatment were measured， followed by the safety evaluation. Result:The PRI， VAS， and PPI scores in SF-MPQ of the observation group were reduced in contrast to those in the control group （<italic>P</italic><0.01）. The wind-cold and stasis obstruction syndrome and ODI scores in the observation group also declined as compared with those of the control group （<italic>P</italic><0.01）， while the ADL and Schober's test scores were elevated （<italic>P</italic><0.01）. The TNF-<italic>α</italic>， IL-1<italic>β</italic>， IL-6， and TXB₂ levels in the observation group were lower than those in the control group （<italic>P</italic><0.01）， whereas the 6-keto-PGF₁<italic>_α</italic> was higher （<italic>P</italic><0.01）. The excellent and good rate of the observation group was （51/63） 80.95%， significantly better than （39/63） 61.90% of the control group （<italic>χ</italic>²＝5.601， <italic>P</italic><0.05）. No adverse reactions occurred after the intake of Chinese medicinal prescriptions. Conclusion:The modified Shentong Zhuyutang combined with celecoxib capsules is efficient and safe in inhibiting inflammation， easing back pain， enhancing lumbar spine mobility， and improving the ADL of patients with acute DLBP of wind-cold obstruction syndrome.

Simultaneous Determination of Eight Active Components in Material Benchmark of Shentong Zhuyutang by HPLC-DAD Wavelength Switching / 中国实验方剂学杂志

Objective::To develop high performance liquid chromatography-diode array detector (HPLC-DAD) wavelength switching for simultaneously determining the contents of inosine, loganic acid, chlorogenic acid, amygdalin, hydroxysafflor yellow A, gentiopicroside, ferulic acid and liquiritin in 15 batches of material benchmarks of Shentong Zhuyutang. Method::The quantitative analysis was carried out on a Thermo Hypersil GOLD C18 column (4.6 mm×250 mm, 5 μm) with mobile phase of acetonitrile-0.1%phosphoric acid aqueous solution for gradient elution, the flow rate was 1.0 mL·min-1, the detection wavelengths were set as 248 nm (0-11 min, inosine), 235 nm (11-14 min, loganic acid), 324 nm (14-16 min, chlorogenic acid), 220 nm (16-19 min, amygdalin and hydroxysafflor yellow A), 274 nm (19-26 min, gentiopicroside), 247 nm (26-54 min, ferulic acid and liquiritin), the column temperature was maintained at 25 ℃. According to the contents of eight active components in 15 batches of material benchmarks, orthogonal partial least squares discriminant analysis (OPLS-DA) in SIMCA 14.1 was used to evaluate the quality difference of each batch of samples. Result::Each component had good separations, the linear ranges of the above 8 components were 2.1-67.2, 1.812 5-58, 1.937 5-62, 5.212 5-166.8, 8.45-270.4, 7.075-226.4, 1.775-56.8, 3.875-124 mg·L-1, respectively (r≥0.999 6). The average recoveries of them were 99.23%, 100.09%, 99.33%, 98.85%, 99.15%, 98.75%, 99.42%, 98.96%, respectively (RSD<2%). The contents of the above eight components in 15 batches of material benchmarks were 0.183 5-0.250 3, 0.173 1-0.265 3, 0.069 5-0.169 8, 0.959 2-1.458 2, 1.905 4-2.553 3, 0.933 3-1.997 5, 0.084 6-0.143 4, 0.212 5-0.704 3 mg·g-1, respectively. Liquiritin, ferulic acid, gentiopicroside and hydroxysafflor yellow A were determined to have significant impact on the quality of different batches of material benchmarks of Shentong Zhuyutang through OPLS-DA. Conclusion::The established method for simultaneous determination of multi-components is reliable, simple and in line with the requirements of methodological verification. It is suitable for the quality control of research and development of compound preparations of Shentong Zhuyutang.

Clinical Observation of Addition and Subtraction Therapy of Shentong Zhuyutang to Myofascial Pain Syndrome (MPS) with Stagnation of Blood Stasis / 中国实验方剂学杂志

Objective: To observe the short and long term effect of addition and subtraction therapy of Shentong Zhuyutang to myofascial pain syndrome (MPS) with stagnation of blood stasis and to investigate its mechanism of action.Method: One hundred and forty-eight eligible patients were randomly divided into control group (73 cases) and observation group (75 cases) by random number table.Patients in both groups got electroacupuncture treatment.Patients in control group additionally got Yaotong capsules,4 grains/time,3 times/day.Patients in observation group additionally got addition and subtraction therapy of Shentong Zhuyutang,1 dose/day.The treatment was continued for 6 weeks in both groups,and 16 weeks follow-up was recorded.Before treatment,and at the 1st,2nd,3rd,4th,5th,6th week after treatment,scores of visual analogue scale (VAS) was graded.Before and after treatment,scores of JOA,Roland-Morris disability questionnaire (RDQ),body damage index assessment scale (PⅡ scale),stagnation of blood stasis,and Pittsburgh sleep quality index (PSQI) were graded.Levels of thromboxane 2(TXB2),6-ketone-prostaglandin F1α(6-keto-PGF1α) were detected,and TXB2/6-keto-PGF1α was calculated;in addition,the recurrence was recorded and followed up.Result: By rank sum test,the clinical efficacy in observation group was better than that in control group (Z=1.969,Pst,2nd,3rd,4th,5th,6th week in both groups after treatment (Fcontrol=5.801,Fobservation=6.649,Pt-test indicated that scores of VAS in observation group were lower than those in control group at 2nd,3rd,4th,5th,6th week (PP2,and T/K in observation group were lower than those in control group (P1α was higher than that in control group (Pχ2=4.745,PConclusion: Based on acupuncture treatment,addition and subtraction therapy of Shentong Zhuyutang can relieve the pain caused by myofascial pain syndrome (MPS) with stagnation of blood stasis,recover lumbar activity function,reduce rate of recurrence,and ameliorate blood circulation,with anti-inflammatory and analgesia effects.