Genetic transformation is a fundamental tool in molecular biology research of medicinal plants. Tailoring transgenic technologies to each distinct medicinal plant would necessitate a substantial investment of time and effort. Here, we present a simple hairy root transformation method that does not require sterile conditions, utilizing Agrobacterium rhizogenes strain K599 and the visible RUBY reporter system. Transgenic hairy roots were obtained for six tested medicinal plant species, roots or rhizomes of which have recognized medicinal value, spanning four botanical families and six genera (Platycodon grandiflorus, Atractylodes macrocephala, Scutellaria baicalensis, Codonopsis pilosula, Astragalus membranaceus, and Glycyrrhiza uralensis). Furthermore, two previously identified Glycyrrhiza uralensis UGTs that convert liquiritigenin into liquiritin in heterologous systems were studied in planta using the method. Our results indicate that overexpression of GuUGT1 but not GuUGT10 and Cas9-mediated knockout of GuUGT1 profoundly influenced the accumulation of liquiritin and isoliquiritin in licorice roots. Therefore, the method described here represents a simple, rapid and widely applicable hairy root transformation method that enables fast gene functional study in medicinal plants.

OBJECTIVE To analyze the metabolites of Zhideke granules and speculate its metabolic pathway in rats in vivo. METHODS Male SD rats were randomly divided into blank group and administration group （Zhideke granules， 9.45 g/kg）； they were given ultrapure water or relevant medicine， twice a day， every 6-8 h， for 3 consecutive days. Serum， urine and feces samples of rats were collected， and their metabolites were identified by UPLC-Q-Exactive-MS technique after intragastric administration of Zhideke granules； their metabolic pathways were speculated. RESULTS After intragastric administration of Zhideke granules， 16 prototype components （i.g. irisflorentin， baicalin， chlorogenic acid） and 11 metabolites （i.g. hydration products of kaempferol or luteolin， methylation products of chlorogenic acid， and hydroxylation products of baicalin） were identified in serum， urine and feces of rats. Among them， 8 prototype components and 4 metabolites were identified in serum samples； 10 prototype components and 7 metabolites were identified in urine samples； 8 prototype components and 5 metabolites were identified in the fecal samples. CONCLUSIONS The metabolites of Zhideke granules in rats mainly include baicalin， irisflorentin，chlorogenic acid， and the main metabolic pathways included methylation， hydroxylation， glucuronidation.

Objective To investigate the clinical effect of transcatheter arterial chemoembolization (TACE) combined with microwave ablation (MWA) (TACE-MWA) versus repeat resection (RR) in the treatment of recurrent hepatocellular carcinoma (RHCC). Methods A total of 178 patients with RHCC who were admitted to The Second People's Hospital of Neijiang from June 2015 to September 2020 were enrolled, and according to the treatment modality, they were divided into RR group with 64 patients and TACE-MWA group with 114 patients. Baseline demographic data, liver function, and tumor conditions before treatment were recorded, and the patients were followed up to October 2021 to compare postoperative overall survival (OS) time and recurrence-free survival (RFS) time between the two groups. Subgroup analysis based on recurrence pattern (recurrence time and tumor size) was performed, and the influencing factors for prognosis were analyzed. The independent samples t -test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data; the Kaplan-Meier method was used for postoperative survival rate, the Log-rank test was used for survival difference analysis, and a multivariate Cox regression analysis was used to investigate independent risk factors for survival. Results The multivariate analysis showed that tumor diameter, alpha-fetoprotein (AFP) level, alanine aminotransferase, albumin, and time to recurrence were independent prognostic factors for OS (all P < 0.05), and AFP level and time to recurrence were independent prognostic factors for RFS (both P < 0.05). For RHCC with late recurrence (> 2 years), there were significant differences between the two groups in median OS (54.0 months vs 36.0 months, χ 2 =6.171, P =0.013) and median RFS (28.0 months vs 21.0 months, χ 2 = 5.211, P =0.022). For RHCC with a tumor diameter of ≤5 cm, there was a significant difference in median OS between the two groups (33.0 months vs 27.0 months, χ 2 =6.447, P =0.011). Conclusion RR has a similar clinical effect to TACE-MWA in RHCC with early recurrence or a tumor diameter of > 5 cm, but RR should be the first choice for RHCC with late recurrence or a tumor diameter of ≤5 cm.

Objective To investigate the influence of preoperative serum heat shock protein 90α (HSP90α) level on the survival time of patients with hepatocellular carcinoma treated by transarterial chemoembolization (TACE). Methods A retrospective analysis was performed for the clinical data of 97 patients with hepatocellular carcinoma who received TACE alone in Department of Hepatobiliary Surgery, The Affiliated Hospital of Southwest Medical University, from January 1, 2019 to June 1, 2020. With the median of serum HSP90α level as the cut-off value, the patients were divided into high-level group with 48 patients (HSP90α > 135 ng/L) and low-level group with 49 patients (HSP90α ≤135 ng/L). The chi-square test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to calculate the median survival time, and the log-rank test was used for comparison between groups. The log-rank univariate analysis and multivariate Cox regression analysis were used to explore the influencing factors for the survival time of patients after surgery. Results There were significant differences between the high-level group and the low-level group in Child-Pugh class ( χ 2 =19.356, P <0.01), tumor necrosis ( χ 2 =9.964, P =0.002), BCLC staging ( χ 2 =22.356, P <0.01), and ECOG score ( χ 2 =6.644, P <0.05). The high-level group had a significantly shorter median survival time than the low-level group ( χ 2 =15.551, P <0.01). HSP90α level (hazard ratio [ HR ]=1.690, P <0.05) and BCLC staging ( HR =2.373, P <0.05) were independent influencing factors for the survival time of patients with hepatocellular carcinoma after TACE. Conclusion Preoperative serum HSP90α level is an independent influencing factor for the survival time of patients with hepatocellular carcinoma after TACE, and it is expected to become one of the potential indicators for evaluating the prognosis of patients with hepatocellular carcinoma treated by TACE.

OBJECTIVE：To optimize the extraction technology of Zhideke granules. ME THODS：The extraction technology （water extraction ，alcohol extraction ，water extraction and ethanol precipitation ）of Zhideke granules was initially screened by ammonia-induced cough experiment and xylene-induced ear swelling experiment in mice. Based on its preparation route ，the immersion time of medicinal materials containing volatile oil was investigated with water absorption as index firstly. The single factor test was adopted to investigate the amount of water added and the extraction time taking the volatile oil yield as index to optimize the extraction technology of medicinal materials containing volatile oil. Taking the contents of irisflorentin and total flavonoids as indicators ，on the basis of single factor investigation ，orthogonal test was adopted to examine the influence of three factors including the amount of water added ，extraction time and extraction frequency ，so as to optimize the water extraction technology of Zhideke granules and the validation tests were conducted. RESULTS ：The results of pharmacodynamics experiment showed that the cough latency of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug ）and water-extraction alcohol-precipitation extract high-dose group （12.68 g/kg，by crude drug ）were significantly longer than those inmodel group ，and the number of cough within 2 minutes was significantly reduced （P＜0.05 or P＜0.01）. Compared with model group ， the ear swelling of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug），ethanol extract high-dose group （12.68 g/kg，by crude drug） and water-extraction alcohol-precipitation extract hig dose group （12.68 g/kg，by crude drug ） were decreased significantly （P＜0.05 or P＜0.01）. The swelling inhibition rates were 42.26％，55.08％，33.49％，51.56％，39.57％ and 44.36％ in low-dose and high-dose groups of water extract ，alcohol extract ， water-extraction and alcohol-precipitation extract respectively ，indicating that the water extract had better antitussive and anti-inflammatory effects. The optimal extraction technology of volatile oil was adding 5-fold water ，soaking for 30 minutes，and extracting for 3 hours. The optimal water extraction technology was adding 12-fold water ，extracting for 3 times after soaked for 50 min，lasting for 1 h each time. Results of 3 times of validation tests showed that average content of irisflorentin in the extract obtained by optimal technology was 76.47 μg/g（RSD＝ 2.15％，n＝3）and the average content of total flavonoids was 92.45 mg/g（RSD＝0.48％，n＝3）. CONCLUSIONS ：The optimal extraction technology of Zhideke granules is stable and feasible.

OBJECTIVE：To establish HPLC fingerprints of different polar parts of Zhuang medicine Calonyction muricatum ， and to study its spectrum-effect relationship with analgesic and anti-inflammatory effects. METHODS ：The total part ，ethyl acetate part，n-butanol part and water part of C. muricatum were prepared. HPLC fingerprints of different polar parts were established by HPLC method combined with the Similarity Evaluation System of TCM Chromatogramtic Fingerprint （2012A），and the common peaks were identified. Using writhing times and ear swelling degree in mice as analgesic and anti-inflammatory indexes ，analgesic and anti-inflammatory activity of different polar parts of C. muricatum were investigated. The correlation of the common peaks of HPLC fingerprint with analgesic and anti-inflammatory indexes was analyzed by grey correlation analysis ，bivariate correlation analysis and partial least square （PLS） method. RESULTS ： There were 11 common peaks for the different polar parts of C. muricatum ，and 5 components were identified by reference comparison，i.e. neochlorogenic acid （peak 3），chlorogenic acid （peak 5）， cryptochlorogenic acid （peak 6）， isochlorogenic acid A （peak 10），isochlorogenic acid C （peak 11）. The grey correlation analysis showed that the correlation between all common peaks and analgesic and anti- inflammatory effects were greater than 0.6 （except the correlation between peak 6 and analgesic effects ），showing correlation relationship ；the correlation of peaks 3，7 and 10 with analgesic and anti-inflammatory effects were all greater than 0.8，which was highly related. Bivariate correlation analysis showed that the correlation of peak 1，3，4，7，9，10，11 with analgesic and anti-inflammatory effects were all greater than 0.6，showing correlation relationship. PLS method showed that peaks 1，3，4，7，9，10，11 contributed greatly to playing an analgesic and anti-inflammatory role. CONCLUSIONS ：HPLC fingerprints of different polar parts of C. muricatum is established and five common peak components were identified. Neochlorogenic acid ，isochlorogenic acid A ，isochlorogenic acid C and chemical components represented by peaks 1，4，7，9 may be the pharmacodynamic substances of C. muricatum to exert analgesic and anti-inflammatory effects.

OBJECTIVE：To i nvestigate the spectrum-effect relationship of analgesic and anti-inflammatory effects of ethyl acetate extract from Zhuang medicine Stahlianthus involucratus from different habitats. METHODS ：Ten batches of S. involucratus from different habitats were used as samples to investigate the anti-inflammatory and analgesic activities of ethyl acetate extracts by xylene induced ear swelling test and acetic acid induced writhing test in mice. HPLC fingerprints of 10 batches of ethyl acetate extract from S. involucratus were established and their similarity was evaluated by using Similarity Evaluation System of TCM Chromatogram Fingerprint （2012 edition），and the common peaks were identified by comparison with the control. The spectrum-effect relationship of anti-inflammatory and analgesic effects of ethyl acetate extract from S. involucratus were analyzed on the basis of Pearson correlation coefficient （auricle swelling degree and writhing times in 15 min as pharmacodynamic indexes ）and Grey relational analysis （inhibition rate of ear swelling and analgesic rate as pharmacodynamic indexes ）. RESULTS ： batches of ethyl acetate extract from S. involucratus had obvious anti-inflammatory and analgesic effects ； inhibition rates of ear swelling in mice were 46.43％-55.16％，and the analgesic rates of mice were 45.56％-52.72％. A total of 18 common peaks were identified in 10 batches of samples ，andthe similarity between them and the control fingerprint was 0.994-0.997. Compared with substance control ，the pea ks 1，2 and 4 were identified as protocatechuic acid ， p-hydroxy- 0771-4953513。E-mail：liangjie1101@126.com benzoic acid and p-hydroxybenzaldehyde，respectively. Results of Pearson correlation analysis showed that peak 10 and peak 18 were significantly negative correlated with auricle swelling degree and writhing times in 15 min（r were values －0.853，－0.738，P values were 0.002，0.015，respectively）. Results of Gray correlation degree analysis showed that the correlation degree of 18 common peaks with inhibition rate of ear swelling and analgesic rate were all greater than 0.65；among them ，peaks 14，1（protocatechuic acid ），17，9，4（p-hydroxybenzaldehyde），2（p-hydroxybenzoic acid ）， 16，7 and 6 showed the relatively high correlation degree （correlation degree ＞0.7）；peak 1（protocatechuic acid ），17，14，9，16，2 （p-hydroxybenzoic acid ）and 4（p-hydroxybenzaldehyde）showed the relatively high correlation degree （correlation degree ＞0.7）. CONCLUSIONS：The ethyl acetate extract of S. involucratus show good anti-inflammatory and analgesic effects. Peak 1 （protocatechuic acid ），2（p-hydroxybenzoic acid ），10，14，17，18 may be its main active ingredients.

Objective To explore the risk factors of dysphagia in the patients with traumatic brain injuries (TBI). Methods 141 TBI patients were assessed with Gugging Swallowing Screen, and divided into the dysphagia (n=36) and normal (n=105) groups. The related factors were compared between 2 groups. Results The frequency increased in severe brain injury, brain stem injury, bilateral brain injury, tracheotomy, nasal-gastric tube insertion and cognitive defect in the dysphagia group compared with those in the normal group (P<0.05). Conclusion The severe brain injury, brain stem injury, bilateral brain injury, tracheotomy, nasal-gastric tube insertion and cognitive defect may be related with the dysphagia post TBI.

Objective To observe the effect of cognitive rehabilitation training based on games on cognition of the traumatic brain injury (TBI) patients.Methods 60 cases of patients according with the inclusion and exclusion criteria were divided into the control group and the training group with 30 cases in each group.The Montreal cognitive assessment (MoCA) test scores of all patients were lower than 26 points.The training group accepted the cognitive rehabilitation training based on games such as exactly the same game,drum game and picture memory game,while the control group did not accept the training.After one month,all patients accepted the MoCA test again.Then we analyzed the differences of the cognition between the two groups.Results After 30 days of training,all items of the cognitive function increased except the sub-item of abstraction.While in the control group,only the scores of attention,delayed recall,orientation and the total score showed alleviation.And all the scores of the training group were higher than those of the control group except the sub-item of abstraction.Conclusions Cognitive rehabilitation training based on games can effectively improve the cognitive function of TBI patients.

Objective To compare the difference between GUSS swallowing evaluation and traditional evaluation as a symbol of pulling out stomach tube.Methods 60 patients with cerebral hemorrhage combined with indwelling tube were divided into two groups,the GUSS group and the traditional group,according to the symbol of pulling out the gastric tube.Each group contained 30 cases.The GUSS score ≥ 15 points was used as the symbol of pulling out the gastric tube in the GUSS group,while being able to eat 200 ml liquid without any trouble in two days was used as the symbol in the traditional group.Then we compared the incidence of gastric tube reinsertion and aspiration pneumonitis between the two groups.Results No gastric tube reinsertion and aspiration pneumonitis occurred in the GUSS group,while 7 cases accepted gastric tube reinsertion and 3 cases diagnosed as aspiration pneumonitis in the traditional group.The incidence of gastric tube reinsertion and aspiration pneumonitis in the GUSS group were obviously lower than those in the traditional group.Conclusions GUSS evaluation score ≥ 15 points could be used as a symbol of pulling out gastric tube.