ObjectiveTo observe the effects and underlying mechanisms of Fagopyri Dibotryis Rhizoma extract on the proliferation， apoptosis， and autophagy of human colorectal cancer HCT-116 cells. MethodFirstly， cellular activity was detected by the cell proliferation and activity-8 （CCK-8） assay， and the half inhibition rate （IC₅₀） was calculated. Blank group and Fagopyri Dibotryis Rhizoma group （2， 4， 8 mg·L^-1） were set. The effect of Fagopyri Dibotryis Rhizoma on the proliferation of HCT-116 cells was observed by cloning experiments， and that of Fagopyri Dibotryis Rhizoma on apoptosis was observed by flow cytometry. The expressions of autophagy-related proteins， p38 mitogen-activated protein kinase （p38 MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun amino-terminal kinase （JNK）， phosphorylated （p）-p38， p-ERK， p-JNK， and other proteins were detected by Western blot. Finally， flow cytometry instrumentation and fluorescence microscopy were used to analyze the changes in reactive oxygen species （ROS）， and a ROS scavenger （NAC） was adopted for verification. ResultCompared with the blank group， the activity of HCT-116 cells was significantly decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）. The apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma group was significantly increased （P<0.01）. The expression of autophagy-related protein ubiquitin-binding protein （p62） was decreased， but that of autophagy-specific genes （Beclin1） and autophagic microtubule-associated protein 1 light-chain 3B （LC3B） was enhanced （P<0.05， P<0.01）. Compared with the Fagopyri Dibotryis Rhizoma group， the apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma + NAC group was significantly reduced （P<0.01）. The expression of related autophagy protein Beclin1 was significantly reduced （P<0.01）， and that of LC3B protein was reduced （P<0.01）. In addition， the expression of MAPK pathway-related proteins ERK and JNK was decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）， and that of p-ERK and p-JNK was enhanced （P<0.05， P<0.01）. ConclusionFagopyri Dibotryis Rhizoma can inhibit the proliferation of HCT-116 cells and induce apoptosis and autophagy through the ROS/MAPK signaling pathway.

【Objective】 To investigate the effects of different anticoagulants on platelet-rich plasma(PRP) release content of growth factor and injection pain. 【Methods】 A total of 15 voluntary blood donors were selected, with each blood donor using four kinds of anticoagulant tubes with EDTA-K2 anticoagulation, EDTA-NA2 anticoagulation, citrate anticoagulation, ACD-A anticoagulation respectively as group A, B, C and D. PRP was isolated and prepared by the rich plasma method, and the contents of PDGF-AA, TGF-β, IGF-1, VEGF, and PF-4 were detected by enzyme-linked immunosorbent assay. Meanwhile, SD rats (20, 4 / group) were injected subcutaneously or intradermally with the supernatant of PRP and PG gel prepared in the 4 groups and normal saline in the control group. The pain status of SD rats during the injection was observed and recorded. The pain status of the 5 groups of experimental animals was evaluated according to the American Laboratory Animal Pain Guide. 【Results】 The platelet counts in PRP in group D was the highest [(1 294.53±277.37) × 10⁹/L], which was significantly higher than that in group A [ (789.13±377.13) ×10⁹/L] and group C [ (990.94±493.12) ×10⁹/L] (P<0.05). The OD value of PDGF-AA in group A, B, C, and D were 1.51± 0.18, 1.69±0.21, 0.66±0.19and 1.72±0.13, respectively, with statistically significant difference between groups (P<0.05 ) and group D better than the other three groups. The OD value of PF-4 was 1.18±0.24, 1.61±0.14, 0.65±0.26 and 1.72±0.10 respectively, with statistically significant difference between groups (P<0.05) and group D better than other three groups. The OD value of IGF-1 was 1.02±0.08, 0.98±0.11, 1.06±0.11 and 1.32±0.65 respectively, with no significant difference between groups (P>0.05). The OD value of VEGF was 0.13±0.04, 0.21±0.14, 0.08±0.02 and 0.13±0.04 respectively, with statistically significant difference between group B and C (P<0.05). The OD value of TGF-β was 0.14±0.01, 0.15±0.01, 0.28±0.17 and 1.10±0.37 respectively, with statistically significant difference between groups (P<0.05) and group D better than other three groups. Comparison of injection pain: when the supernatant of PRP and PG gel was injected, there were significant differences between group A, B, C and D, and the control group (P<0.05) . The median pain scores of PRP injection of group A, B, C, and D were 6 (1.5), 5 (0.75), 4.5 (2.5), and 3(3) respectively, with group D lower than other three groups, and no statistically significant difference was noticed (P>0.05) . The median pain scores of the PG supernatant injection of group A, B, C, and D were 4 (2.25), 3 (2.75), 4 (3), 1 (1.5), and the difference was not statistically significant (P>0.05). There was no significant difference between the PRP injection group and the PG supernatant group (P> 0.05). 【Conclusion】 PRP prepared by two-step centrifugation with ACD-A anticoagulant can obtain the higher platelet counts and the maximum release of PDGF-AA, PF-4, IGF-1, and TGF-β. In terms of pain, ACD-A anticoagulant injection has the lowest pain with the animals.

【Objective】 To develop a spray-on membrane dressing for wound repair containing platelet rich plasma (PRP) sodium alginate (SA)/agarose(AG)/carboxymethyl chitosan (CMCS). 【Methods】 SA/AG/ CMCS were mixed in different proportions to prepare biodegradable quick setting spray (BQSS) by blending film method, and the film-forming time, moisture retention and compression resistance of the prepared BQSS were tested. Then PRP and BQSS were mixed in the proportion of 3∶7, 4∶6, 5∶5, 6∶4 and 7∶3 to prepare PRP-BQSS spray film dressings. The film-forming time, moisture retention, compressive strength, porosity and slow-release effect of growth factors of PRP-BQSS spray film dressings were studied. 【Results】 In the preparation of BQSS compound spray film solution, when SA, AG, CMCS and sterile distilled water were 0.6∶0.6∶0.6∶98.2g, the film-forming time (7.73±0.31) s, moisture retention (75. 54±3.03) % and compression resistance (791.00±68.02) g of the spray-film dressing were the best. The basic properties of PRP-BQSS spray-on film dressings and the release of growth factors show that PRP-BQSS spray-on film dressings can exist in different forms, and with the decrease of PRP concentration percentage, its film-forming time, moisturizing performance and compressive strength showed an upward trend. When the PRP content is 30%, the porosity of the dressing is the highest, about(84.34±0.90)%. The release of platelet-derived growth factor-AA(PDGF-AA), platelet factor-4(PF-4) and transforming growth factor beta (TGF-β) was in a slow upward trend, and the release of the three growth factors was higher than that of PRP group in 48 hours. 【Conclusion】 The preparation method of PRP-BQSS spray film dressing designed in this study is simple and mild, and can form a film quickly, with good biological properties and better growth factor inhibition and sustained-release effect.

【Objective】 To discuss the effect of temperature on the quality of platelet-rich plasma (PRP) prepared manually. 【Methods】 A total of 120 peripheral blood samples (60 mL/ person) were collected from healthy voluntary blood donors in the Blood Center of General Hospital of Southern Theater Command from May 10, 2010 to September 10, 2010. Each whole blood sample (60mL/ person) was randomly divided into 3 aliquots(20 mL each), totaling 360 aliquots, then divided into 9 groups (A1, A2, A3, B1, B2, B3, C1, C2, and C3), with 40 aliquots in each group. In group A: the ambient temperature for PRP preparation was set to (4±2)℃, and the temperature for PRP preparation and centrifugal bin was (4±2)℃, (24±2)℃ and (30±2)℃ for group A1, A2, and A3, respectively. In group B: the ambient temperature for PRP preparation was set to (24±2)℃, and the temperature for PRP preparation and centrifugal bin was (4±2)℃, (24±2)℃ and (30±2)℃ for group B1, B2 and B3 respectively. In group C: the ambient temperature for PRP preparation was set to (30±2)℃, and the temperature for PRP preparation and centrifugal bin was (4±2)℃, (24±2)℃ and (30±2)℃ for group C1, C2 and C3 respectively. Platelet concentrates were separated and prepared by rich slurry method, and the platelet recovery rate was calculated for each group, the platelet morphology was observed under the microscope, and the concentrations of platelet-derived growth factor (PDGF-BB), transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) were quantitatively determined using enzyme-linked immunosorbent assay (ELISA). 【Results】 The platelet count and platelet recovery rate were compared as follows: among group A1, A2, A3, B1, B2, B3, C1, C2 and C3, the results of A3, B3 and C3were (489.2±21.47) × 10⁹/L vs (495.7±23.2) ×10⁹/L vs (489.4±17.1) ×10⁹/L and (57.4±2.3)% vs (54.9±1.3)% vs (50.7±2.3)%, respectively, all showed platelet aggregation, and the differences, relative to A1 and A2, B1 and B2, and C1 and C2, were statistically significant (P<0.05). Microscopic observation was as follows: in the PRPs collected from group A1, A2, B1, B2, C1 and C2, the platelet cells were uniform in size and without aggregation; in the PRPs collected from group A3, B3 and C3, some platelets showed aggregation. Determination of growth factor content was as follows: the content of TGF-β(ng/L) and PDGF-BB(ng/L) was 375.0±119.1 vs 183.67±106.2 and 933.0±273.0 vs 656±113.0 in the non-aggregation group and the aggregation group, respectively (P<0.05), while the content of VEGF(ng/L) was 217.5±93.5 vs 155.3±103.4 (P>0.05), with no statistically difference. 【Conclusion】 The ambient temperature had little effect on the preparation of PRP, and the temperature of the centrifuge bin was better to be maintained at (24±2)℃.

【Objective】 To analyze the influencing factors of clinical efficacy of autologous platelet-rich plasma in the treatment of androgenetic alopecia. 【Methods】 From January, 2019 to December, 2021, 151 outpatients with androgenetic alopecia were treated in our hospital. All patients were treated with autologous PRP injection once a month and planned to received injection for 4-6 occasions. According to the hair growth before and after treatment, the treatment results were evaluated. The influencing factors in the treatment results were grouped and analyzed, including the treatment numbers (divided into 1-6 occasions), the grade of alopecia(three grades: mild, moderate and severe alopecia) and the Plt concentration in PRP[five grades(×10⁹/L): extremely low concentration (<800), low concentration (801-1 000) and medium concentration (1 001-1 200), high concentration(1 201-1 500)and extremely high concentration(>1 501)]. 【Results】 The correlation coefficient between the number of treatments and the effective rate was pearson=0.986, P<0.001, showing a positive correlation. There was a statistical difference in the total number of treatments between groups (P<0.01). As to the severity of alopecia: The effective rates of mild and moderate alopecia patients (66.67%, 56.95%) were higher than those of severe alopecia patients (35%). The difference between groups was statistically significant (P<0.01). As to the concentration of Plt in PRP: The concentration of Plt directly affected the therapeutic effect. When the Plt count was (1 200-1 500)×10⁹/L, the effect was optimal, reaching 68.12%, which was significantly higher than the other four groups(P<0.01). The treatment numbers, alopecia grade and Plt concentration in PRP were all related to the effectiveness of treatment (P<0.05). When the OR value of treatment numbers and Plt concentration was >1, the effective rate increased by 2.619 times and 2.033 times, respectively, as treatment numbers and Plt concentration increased. When the OR value of the grade of alopecia was 0.338 (0<0.338<1), the effective rate decreased with the increase of alopecia severity; For each grade increased, the effective rate would be compromised by 0.338. 【Conclusion】 When PRP is applied to treat AGA patients, the clinical therapeutic effect will be better in patients who received more treatments, PRP with higher Plt count, and be in the early degree of hair loss. Whether the higher the concentration of Plt in PRP, the better the therapeutic effect will be, remains to be further verified.

【Objective】 To discuss the repair effect of lyophilized platelet lysate (PL) products on articular cartilage injury model of rats. 【Methods】 A total of 25 SD rats were injected with typeⅡcollagenase at the right knuckle articular cavity respectively on day 1, 3 and 5 of experiment, and the modeling conditions were observed 14 days after the last injection of collagenase. The SD rats with successful modeling were randomly divided into three groups, and were injected with lyophilized PL [Group A, 1 mL/(mouse·time)], PL [Group B, 1 mL/(mouse·time)], and normal saline[Group C, 1 mL/(mouse·time)]. The above three substances were injected with corresponding drugs on day 0, 7, 14 and 21 of experiment based on the grouping conditions, and the changes of knee joint diameters of the rats from the three groups were observed and compared. On day 14 and 28, one rat in each group was randomly killed and two knuckle articular cavities of each were taken for tissue sampling, using hematoxylin-eosin staining (HE) and immunohistochemistry. 【Results】 After 14 days of modeling by injection of type Ⅱ collagenase, the proportion of successful modeling in rats was 84% (21/25), with the knee joint diameter (mm) before and after modeling at 12.84±1.14 vs 14.11±1.17(P<0.01). On day 14, 21 and 28, groups A and B were superior to group C in the knee joint diameter and activity improvement (P<0.05), with 13.33±1.16 vs 13.37±1.08 vs 14.21±1.08, 13.10±1.09 vs 13.01±1.04 vs 14.09±1.09 and 12.38±1.08 vs 12.51±1.03 vs 14.01±1.07, respectively. Histological observation showed that group A and B were superior to group C in the production and arrangement of chondrocytes and the positive expression of type Ⅱ collagen, and there was no significant difference between group A and group B. 【Conclusion】 The lyophilized PL has similar therapeutic effect to PL in the treatment and repair of articular cartilage injury, and is worthy of clinical application.

【Objective】 To explore the clinical effects of autologous platelet-rich plasma (PRP) in the treatment of postoperative bone nonunion for long bone shaft fractures during operation and after operation. 【Methods】 A total of 34 patients with postoperative bone nonunion for long bone shaft fracture, who were admitted and cured in the Orthopedics Department of our hospital from January 2019 to June 2020, were selected. They were randomly divided into the experimental group(n=17) and control group(n=17). Individuals in the control were treated with autologous iliac bone alone., while the experimental group were treated with autologous iliac bone graft plus autologous PRP during and after operation. After surgery, the autogenous PRP was accurately injected with ultrasound guidance every 7 days (5 mL/person, 4 occasions in total). The clinical healing time of the fracture, the grading of callus formation and the functional rehabilitation level of limbs on the affected side at different time were observed in both groups. 【Results】 All 34 patients were followed up. In the experimental group and the control group, the clinical healing time (month) of the fracture was (5.03±1.24) vs (6.91±1.41), P<0.05. The healing rate of the fracture for 6 months and 9 months was 94%(16/17)vs 59%(10/17)and 94%(16/17)vs 82%(14/17), respectively (P<0.05). The grading of callus formation within 3, 6 and 9 months was (2.11±0.69) vs (1.53±0.80), (3.06±0.90) vs (2.59±0.87) and (3.82±0.73) vs (3.35±0.86), respectively (P<0.05). The acceptance rate of functional rehabilitation of limbs on the affected side was 82.35%(14/17)vs 76.47%(13/17), P<0.05. 【Conclusion】 The application of autologous PRP in the treatment of postoperative bone nonunion for long bone shaft fractures during operation and after operation can achieve good clinical treatment.

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.
Biocatalysis ; Biotechnology ; Catalysis ; Drug Compounding ; Enzymes/metabolism*

Objective To investigate the expression and significance of inducible nitric oxide synthase (iNOS), platelet-derived growth factor (PDGF)-B and lipopolysaccharide (LPS) in rat models with Budd-Chiari syndrome (BCS) . Methods BCS model was established by partial ligation of inferior vena cava in the posterior segment of the liver. The experimental rats were divided into control group (n=20), model group (n=20) and sham group (n=20) . Liver tissues were collected for immunohistochemistry, HE and Masson staining, and the expression levels of iNOS, PDGF-B and LPS were determined. Results The LPS value in model group was higher than that in both control group and sham group (P=0.001) . The mRNA and protein expressions of iNOS and PDGF-B in model group were higher than those in both control group and sham group (P=0.001) . Statistically significant differences in mRNA and protein expressions of iNOS and PDGF-B existed between each other among the subgroups (P=0.001) . In model group iNOS was positively correlated with PDGF-B and LPS; liver fibrosis was positively correlated with LPS and negatively correlated with PDGFB. Conclusion The damage and repair of BCS is a complicated process. The iNOS, PDGF-B and LPS may play different roles in different stages of BCS. How to regulate their balance in liver fibrosis may be a direction that deserves further study.

Objective:To prepare mesalazineβ-cyclodextrin inclusion compound and observe its properties. Methods:The struc-ture was characterized by UV, IR and DSC. The inclusion process of mesalazine andβ-cyclodextrin was simulated using molecular doc-king technique. The solubility and dissolution of inclusion compound were determined. Results:UV, IR and DSC were used to deter-mine the formation of inclusion complex. The inclusion ratio was 1: 1 and the phase solubility diagram was AL type. The solubility of inclusion compound in deionized water (25 ℃, pH 7. 0) was 7. 8 mg·ml-1 . The preparation of mesalazine β-cyclodextrin inclusion compound could significantly increase the dissolution rate and solubility of mesalazine. Conclusion:The prepared mesalazineβ-cyclo-dextrin inclusion compound can obviously improve the poor water solubility and dissolution of mesalazine.