ObjectiveTo investigate the antitumor effect and mechanism of Guiqi Yiyuan ointment on Lewis lung cancer mice based on the extracellular regulatory protein kinase （ERK） signaling pathway. MethodsA Lewis lung cancer mouse model was established. Except for the blank group， the model mice were randomly divided into the model group， Guiqi Yiyuan ointment low， medium， and high dose groups， and the extracellular ERK1/2 inhibitor group， with 10 mice per group. The Guiqi Yiyuan ointment was administered by gavage at doses of 1.75， 3.5， 7.0 g·kg^-1·d^-1 for the low， medium， and high dose groups， respectively. The ERK1/2 inhibitor group was given the ERK1/2 inhibitor LY3214996 （100 mg·kg^-1·d^-1） by gavage. The treatment was administered for 14 consecutive days， after which samples were collected. Tumor histopathological changes were observed using hematoxylin-eosin （HE） staining. Transmission electron microscopy was used to observe ultrastructural changes in tumor cells. Immunofluorescence was performed to measure the phosphorylation of ERK1/2 （p-ERK1/2） and the expression of programmed cell death ligand-1 （PD-L1） in tumor tissues. Western blot and real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect the expression of p-ERK1/2， PD-L1， the autophagy marker Beclin-1， the autophagic protein p62， and the microtubule-associated protein light chains LC3Ⅰ and LC3Ⅱ at both the protein and gene levels. ResultsCompared with the model group， the average tumor weight was significantly reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and markedly reduced in the high dose and inhibitor groups （P<0.01）. Tumor cells in all treatment groups became progressively irregular， with ruptured nuclei and expanded areas of cell disintegration and necrosis. The number of organellar ablations in tumor tissues increased， and the number of autophagic vesicles also increased in all groups. The mean fluorescence intensity of p-ERK1/2 and PD-L1 was reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and significantly reduced in the high dose and inhibitor groups （P<0.01）. The mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， while LC3Ⅰ/Ⅱ mRNA expression was elevated （P<0.05）. In the high dose and inhibitor groups， mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ mRNA expression was significantly increased （P<0.01）. Protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， and LC3Ⅰ/Ⅱ protein expression was elevated （P<0.05）. In the high dose and inhibitor groups， protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ protein expression was significantly elevated （P<0.01）. ConclusionGuiqi Yiyuan ointment may inhibit the activation of the ERK signaling pathway， downregulate the expression of p-ERK1/2， promote autophagic flux in tumor cells， and regulate the expression of PD-L1， thereby exerting an inhibitory effect on tumor growth in Lewis lung cancer mice.

Objective To investigate the relationship between the expression of long non-coding RNA(LncRNA)small nucleolar RNA host gene 11(SNHG11)and hypoxia inducible factor(HIF)-1α and angiogenesis mimicry(VM)in ovarian cancer.Methods A total of 116 ovarian cancer patients admitted to Tangshan Maternal and Child Health Care Hospital from October 2019 to January 2023 were regarded as the research subjects.Based on whether VM had formed,ovarian cancer patients were grouped into VM group(n=51)and non VM group(n=65).Another 50 partients who underwent health examinations during the same period were regarded as the control group.Real-time fluorescence quantitative PCR(qPCR)was applied to detect the expression levels of LncRNA SNHG11 and HIF-1α in serum of ovarian cancer patients and control groups.Spearman correlation was applied to detect the relationship between LncRNA SNHG11,HIF-1α,and VM formation.The diagnostic value of LncRNA SNHG11,HIF-1α,and their combined detection in the formation of VM in ovarian cancer patients was analyzed using the receiver operating characteristic(ROC)curve.Results Compared with the control group,the levels of serum LncRNA SNHG11(3.01±0.88,2.21±0.68 vs 1.12±0.35)and HIF-1α(2.16±0.67,1.60±0.44 vs 1.01±0.31)in ovarian cancer patients with VM group and non VM group were increased(t=12.136,9.006;19.890,16.591,all P＞0.05),the levels of serum LncRNA SNHG11 and HIF-1αin the VM group were obviously higher than those in the non VM group(t=8.957,8.595),and the differences were statistically significant(all P＜0.05).The expression of LncRNA SNHG11,HIF-1α,and the formation of VM were not related to age and tissue type(t=1.036,0.976,0.218;1.254,1.390,0.368,all P＞0.05),but were related to tumor size,FIGO staging,lymph node metastasis,and pathological grading(t=5.351,5.186,13.264;5.465,5.227,10.898;6.063,6.016,5.374;4.030,5.871,5.509,all P＜0.05).Spearman correlation analysis showed that there were obvious positive correlations between LncRNA SNHG11,HIF-1α,and VM generation(r=0.560,0.494,all P<0.05).ROC curve results showed that the areas under the curve(AUCs)of serum LncRNA SNHG11 and HIF-1α for diagnosing VM formation in ovarian cancer patients were 0.860 and 0.824,respectively,with sensitivity of 80.4%and 75.6%,specificity of 58.9%and 51.9%,respectively.The AUC of VM formation in ovarian cancer patients diagnosed by the combination of the two was 0.941,with sensitivity and specificity were 92.2%and 79.9%,respectively.Conclusion The abnormal expressions of LncRNA SNHG11 and HIF-1α were closely related to the formation of VM in ovarian cancer patients,and both may serve as potential biological indicators for judging VM.

ObjectiveTo explore the protective effects of Dahuang Tangluo pills on early diabetic kidkdey disease （DKD） in db/db mice. MethodEight db/m mice were selected as the control group. Forty male db/db mice were selected and blood samples were collected via tail vein to measure fasting blood glucose （FBG）. Mice with FBG ≥ 16.7 mmol·L^-1， increased urine output， and persistent albuminuria were considered successful in model establishment. After successful modeling， they were randomly divided into a model group， a dapagliflozin group （1.5 mg·kg^-1·d^-1）， and high， medium， and low dose groups of Dahuang Tangluo pills （3.6， 1.8， 0.9 g·kg^-1·d^-1， respectively）， with eight mice in each group. All medication groups were administered orally， while the control and model groups were given an equal amount of distilled water by gavage daily. After continuous administration for 10 weeks， the survival status of the mice was observed， and their body weight， FBG， and kidney function-related indicators were measured. Inflammatory indicators in renal tissues were determined by enzyme-linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy were used to observe the pathological changes in renal tissues in each group. Immunofluorescence was employed to examine the expression of advanced glycation end products （AGEs） and receptors for advanced glycation end products （RAGE） proteins. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were utilized to detect the gene and protein expression levels of AGEs， RAGE， inhibitor of nuclear factor-κB （NF-κB） kinase （IKK）， and NF-κB in the renal tissues of mice in each group. ResultCompared with control group， the model group showed a significant increase in body weight， FBG， serum creatinine （SCr）， urinary microalbumin/urine creatinine ratio （ACR）， total cholesterol （TC）， and triglycerides （TG） （P<0.05）. The levels of intercellular adhesion molecule-1 （ICAM-1）， interleukin-6 （IL-6）， and tumor necrosis factor-alpha （TNF-α） in renal tissues were significantly elevated （P<0.05）. Renal histopathological staining and electron microscopy revealed loose arrangement， gaps， structural disarray， mesangial proliferation， and significant fibrosis in renal tissues. Real-time PCR results showed a significant increase in the expression of RAGE， IKK， and NF-κB genes in renal tissues （P<0.05）. Immunofluorescence results demonstrated a significant increase in the expression of AGEs and RAGE proteins in renal tissues （P<0.05）. Western blot results showed a significant increase in the expression of AGEs， RAGE， IKK， and NF-κB proteins in renal tissues （P<0.05）. After drug intervention， compared with model group， the dapagliflozin group and the high-dose Dahuang Tangluo pills group showed significant reductions in body weight， FBG， SCr， and ACR （P<0.05）， and a significant decrease in TC in mouse serum （P<0.05）， while the high-dose Dahuang Tangluo pills group showed a significant decrease in TG in mouse serum （P<0.05）. All treatment groups showed a significant reduction in ICAM-1， IL-6， and TNF-α in renal tissues （P<0.05）. Renal histopathological staining and electron microscopy showed improved kidney injury， decreased collagen fiber deposition， and reduced mesangial proliferation in all treatment groups. Real-time PCR results showed a significant decrease in the expression of RAGE， IKK， and NF-κB genes in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. Immunofluorescence results demonstrated a significant decrease in the expression of AGEs and RAGE proteins in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. Western blot results showed a significant decrease in the expression of AGEs， RAGE， IKK， and NF-κB proteins in the dapagliflozin group and the high- and medium-dose Dahuang Tangluo pills groups （P<0.05）. ConclusionDahuang Tangluo pills can improve the pathological structure of the kidneys and reduce renal inflammation in DKD mice， possibly through inhibiting the AGEs/RAGE/IKK/NF-κB pathway.

Objective To prepare silymarin phospholipids complex（SM-PC） and investigate its physicochemical properties. Methods On the basis of single-factor tests, the drug-lipid ratio, drug concentration and reaction temperature were selected as the factors of the central composite design and response surface methodology in the preparation of SM-PC by solvent volatilization, and the best process was optimized with the compound rate as the index. And its in vitro dissolution was measured. Results The optimum preparation technology of SM-PC was as follows: acetone was used as compound solvent, the concentration of SM was 8.0 mg/ml, the mass ratio of SM to phospholipid was 1∶1.8, the reaction temperature was 56 ℃ and the recombination rate was（95.15±1.55）% with deviation of less than 3%. The in vitro dissolution test showed that the dissolution of SM-PC was close to 90% in 60 min. The dissolution behavior of main component of silybin was similar to that of silymarin capsules（Legalon ^®）, which was higher than SM-API. Conclusion SM-PC was successfully prepared by central composite design response surface method, which significantly improved the dissolution and laid a foundation for the study of subsequent preparations.

Objective To observe the effect of Jinshui Liujun decoction on airway lesions in mice with COPD and explore its possible mechanism.Methods 48 C57BL/6 mice were randomly divided into blank group,model group,Jinshui Liujun decoction group and NAC group,with 12 in each group.The COPD mouse model was established by intranasal drip of LPS and smoking,and the corresponding drugs were given intragastric administration for 14 days after the model was established.Observe the general condition of the mice,measure the MV,PEF and PIF of the mice with the small animal lung function instrument,semi quantitatively evaluate the inflammation of the lung tissue,the thickness of the alveolar septum and the thickness of the airway wall with HE staining,and observe the airway mucus secretion and goblet cell proliferation with PAS staining.The content of MPO,SA and Urea in BALF was detected by the kit,and the ratio of SA and Urea was calculated.The content of MUC5AC in BALF was detected by ELISA.The levels of ROS,GSH,GSSG and GR in lung tissue were detected with the kit,and the ratio of GSH and GSSG was calculated.The expression level of CFTR mRNA in lung tissue was detected by qRT-PCR.Western blot was used to detect the expression level of CFTR protein in lung tissue.Results Compared with the control group,the growth of mice in the model group was poor.The body weight at each time point during the modeling period decreased(P<0.01),and the indexes of MV,PEF and PIF decreased(P<0.01).The lung tissue pathological score,alveolar septal thickness,airway wall thickness,airway mucus and goblet cell increased(P<0.01).The levels of SA,SA/Urea,MUC5AC and MPO in BALF increased(P<0.01),and the level of Urea decreased(P<0.01),The levels of ROS and GSSG in lung tissue increased(P<0.01),and the levels of GSH,GSH/GSSG,and GR decreased(P<0.01).The expression levels of CFTR mRNA and protein in lung tissue decreased(P<0.01).Compared with the model group,the growth condition of COPD mice improved,the body weight increased at each time point during the modeling period(P<0.05,P<0.01),the indexes of MV,PEF and PIF improved significantly(P<0.01),the pathological score of lung tissue,the thickness of alveolar septa,the thickness of airway wall,airway mucus and goblet cell decreased(P<0.01,P<0.05),and the levels of SA,SA/Urea,MUC5AC and MPO in BALF decreased(P<0.01),And an increase in Urea levels(P<0.01),a decrease in ROS and GSSG levels in lung tissue(P<0.01),and an increase in GSH,GSH/GSSG,and GR levels(P<0.01).The expression levels of CFTR mRNA and protein in lung tissue increased(P<0.01).Conclusion Jinshui Liujun decoction can correct CFTR acquired defects through antioxidant effects to improve airway lesions in COPD.

Objective To explore the inhibitory effect of Guiqi Yiyuan Ointment combined with cisplatin on Lewis lung cancer mice and its mechanism based on the lncRNA H19/miR-19b3p/FTH1 axis.Methods Totally 60 SPF grade male C57BL/6 mice were randomly selected as blank group,and Lewis lung cancer mouse model was replicated in the remaining 50 mice.The modeled mice were randomly divided into model group,cisplatin group,and TCM low-,medium-and high-dosage combined with cisplatin group,with 10 mice in each group.The cisplatin group was given intraperitoneal injection of cisplatin 5 mg/kg,while the TCM low-,medium-and high-dosage combined with cisplatin group were given Guiqi Yiyuan Ointment 1.75,3.5,7.0 g/kg by gavage combined with intraperitoneal injection of cisplatin 5 mg/kg,the blank group and model group were given equal volume of normal saline by gavage for 14 consecutive days.The general condition of mice was observed,the tumor mass of mice was measured,and the tumor inhibition rate,spleen index,and thymus index were calculated,HE staining was used to observe the pathological changes of tumor tissue,and the contents of malondialdehyde(MDA),reduced glutathione(GSH),hydrogen peroxide(H2O2),and ferrous ions(Fe2+)were detected in the tumor tissue,immunohistochemistry and Western blot were used to detect the expression of nuclear factor E2 related factor 2(Nrf2),glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1)and solute carrier family 7 member 11(SLC7A11)protein in tumor tissue,real time fluorescence quantitative PCR was used to detect the expression of lncRNA H19 and miR-19b3p mRNA in tumor tissue.Results Compared with the blank group,the body mass,spleen index and thymus index of mice in the model group were decreased(P<0.05).Compared with the model group,the mental state and tumor histopathological morphology of mice in each administration group were improved to different degrees,the tumor mass of mice was reduced(P<0.05),the spleen index and the thymus index of mice increased(P<0.05),tumor cell lysis,rupture,and decrease in quantity,the contents of MDA,H2O2,Fe2+in tumor tissue increased(P<0.05),while GSH content decreased(P<0.05),the expression of Nrf2,GPX4,FTH1,SLC7A11 protein in tumor tissue decreased(P<0.05),the lncRNA H19 mRNA expression decreased(P<0.05),miR-19b3p mRNA expression increased(P<0.05).Compared with the cisplatin group,the tumor mass of mice in TCM high-dosage combined with cisplatin group reduced(P<0.05),the body mass,spleen index and thymus index increased(P<0.05),the contents of MDA,H2O2,Fe2+in tumor tissue of mice increased(P<0.05),the content of GSH decreased(P<0.05),the expression of Nrf2,FTH1,SLC7A11,GPX4 protein expression decreased(P<0.05),lncRNA H19 mRNA expression decreased(P<0.05),miR-19b3p mRNA expression increased(P<0.05).Conclusion Guiqi Yiyuan Ointment combined with cisplatin can significantly inhibit the tumor of Lewis lung cancer mice,and its mechanism may be related to regulating the expression of ferroptosis molecules related to the lncRNA H19/miR-19b3p/FTH1 axis.

Objective To observe the anti-tumor effect and mechanism of Guiqi Yiyuan Ointment combined with cisplatin in Lewis lung cancer bearing mice based on AMPK/ULK1 signaling pathway.Methods The mice were inoculated with Lewis lung cancer cells to establish a subcutaneous tumor model in the right axillary subcutaneous area,and were randomly divided into blank group,model group,cisplatin group and Guiqi Yiyuan Ointment high-,medium-and low-dosage+cisplatin groups,with 10 mice in each group.The cisplatin group received intraperitoneal injection of 5 mg/kg(once every 7 days)cisplatin,while the Guiqi Yiyuan Ointment high-,medium-and low-dosage+cisplatin groups received gavage of 7.0,3.5 and 1.75 g/kg Guiqi Yiyuan Ointment on the basis of cisplatin group,once a day,for 14 consecutive days.HE staining was used to observe the morphology of tumor tissue,immunohistochemical method was used to detect the protein expression of p-AMPK and p-ULK1 in tumor tissue,transmission electron microscopy was used to observe the number of autophagosomes,RT-PCR was used to detect the expression of p62,Beclin-1,Atg5 and LC3 mRNA in tumor tissue,Western blot was used to detect the expression of Beclin-1,p62,Atg5,LC3 and AMPK/ULK1 signaling pathway proteins in tumor tissue.Results Compared with the model group,the tumor mass of mice in all administration groups decreased significantly(P<0.05);the tumor tissue showed patchy necrotic areas and an increase in the number of autophagosomes,the mRNA expressions of Beclin-1,Atg5,and LC3 increased,the protein expressions of Beclin-1,Atg5,LC3 Ⅱ/Ⅰ,AMPK,p-AMPK,ULK1 and p-ULK1 increased,and the expression of p62 mRNA and protein decreased(P<0.05).Compared with the cisplatin group,the tumor mass of mice in Guiqi Yiyuan Ointment high-,medium-dosage+cisplatin groups decreased(P<0.05),the tumor inhibition rate increased;the expressions of ULK1 protein and Atg5 mRNA in tumor tissue of Guiqi Yiyuan Ointment high-,medium-and low-dosage+cisplatin groups significantly increased(P<0.05),the expressions of Beclin-1 and LC3 mRNA in Guiqi Yiyuan Ointment high-,medium-dosage+cisplatin groups significantly increased(P<0.05),the expressions of Atg5,LC3 Ⅱ/Ⅰ,AMPK,p-AMPK and p-ULK1 protein significantly increased(P<0.05),and the expression of p62 protein significantly decreased(P<0.05),the expression of p62 mRNA in Guiqi Yiyuan Ointment high-dosage+cisplatin group significantly decreased(P<0.05),the expression of Beclin-1 protein significantly increased(P<0.05).Conclusion Guiqi Yiyuan Ointment combined with cisplatin may activate autophagy and inhibit subcutaneous tumor growth in Lewis lung cancer bearing mice through the AMPK/ULK1 signaling pathway.

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo, we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l-arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP.

Objective To evaluate the release characteristics in vitro, pharmacokinetics in rabbits and in vivo-in vitro correlation of silymarin phospholipid complex microporous osmotic pump controlled release tablets（SM-PC MPOP）. Methods The release characteristics of SM-PC MPOP in vitro were detected by HPLC in the artificial gastric fluid. Six beagle dogs were subjected to double cycle cross control, which were given SM-PC MPOP and Legalon（30 mg/kg）. The concentration of silybin in plasma was determined by HPLC and the data were processed by software. Results The cumulative release rate of SM-PC MPOP in vitro was over 85% in 12 h. The pharmacokinetics in beagle dogs showed that SM-PC MPOP and legalon conformed to double compartment first-order absorption model and the pharmacokinetic parameters were obtained: t_max:（3.2±0.4）and（0.9±0.1）h, C_max:（0.298 6±0.068 9）and（0.629 9±0.076 5）μg/ml, AUC_0→24:（2.996 8±0.583 3）and（2.268 9±0.432 8）h·μg /ml. The relative bioavailability of SM-PC MPOP was（162.21 ± 30.82）%. Conclusion SM-PC MPOP could release slowly, which could increase the relative bioavailability significantly. The correlation between the absorption in vivo and release in vitro was fine（r = 0.839 0）.

Objective Based on HMGB1/TLR4/NF-κB pathway,to investigate the effects of Shenqi Yiliu decoction combined with cisplatin on H22 liver cancer tumor mice and the effects of related immune indicators.Methods 50 SPF grade male KM mice,10 mice were taken as blank group by random number table method,and the other 40 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mice model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group(2.5×10-3 g·kg-1),Shenqi Yiliu decoction TCM group(27.03 g·kg-1),and Shenqi Yiliu decoction TCM(27.03 g·kg-1)combined with cisplatin(2.5×10-3 g·kg-1),10 mice in each group were treated for 13 d.Determine tumor suppression rate,spleen index and thymus index;HE observes changes in oncology pathology;streaming cells detect the level of CD4+T,CD8+T cells in the spleen tissue;PT-PCR and WB method detect genes and protein expression related to HMGB1/TLR4/NF-κB signaling pathways in tumor tissues.Results ①Compared with the blank group,the mean body mass and mouse spleen index,thymus index,CD4+ T cell level and CD4+T/CD8+T value were significantly lower and CD8+T cell level was higher in the model group(P<0.05);②Compared with the model group,the mean tumor mass decreased(P<0.05),tumor volume decreased(P<0.05),and body mass increased(P<0.05)in each treatment group,and the spleen index,thymus index,CD4+T cell level and CD4+T/CD8+T ratio increased and CD8+T cell level decreased in both the Chinese medicine group and the combination group,and the treatment effect was significant in the Chinese medicine group(P<0.05),and HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice were reduced,and the effect was significant in the combined group(P<0.05).③Compared with the cisplatin group,HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression were reduced in the tumor tissues of mice in the combination group(P<0.05).④HMGB1,TLR4,MyD88,NF-κB mRNA and protein expression in tumor tissues of mice in the combined group were reduced compared with those in the Chinese medicine group(P<0.05).Conclusion Shenqi Yiliu decoction combined with cisplatin can effectively inhibit tumor growth and improve related immune indexes in H22 hepatocellular carcinoma tumor-bearing mice,and the mechanism may be related to the inhibition of HMGB1/TLR4/NF-κB signaling pathway activation.