To explore the mechanism of remimazolam improving learning and memory ability and brain injury in postoperative cognitive impairment (POCD) mice by regulating NOD-like receptor heat protein domain associated protein 3 (NLRP3).

The C57BL/6J mice were divided into sham group (exposure of the tibia only without truncation), model group (tibia ampution method to construct POCD model), oe-NC group (modeling+15 mg·kg^-1 remimazolam treatment+tail vein injection of 60 nmol·L^-1oe-NC), oe-NLRP3 group (modeling+15 mg·kg^-1 remimazolam treatment+tail vein injection of 60 nmol·L^-1oe-NLRP3), experimental-L, -M, -H groups (after modeling, 5, 10, 15 mg·kg^-1 remimazolam was respectively given intraperitoneal injection), 12 rats per group. For the treated mice, the Morris water maze and step - down test were used to evaluate their learning and memory abilities. The expression levels of NLRP3, interleukin-1β (IL-1β), IL-10 and tumor necrosis factor-α (TNF-α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The level of reactive oxygen species (ROS) was detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and the level of glial fibrillary acidic protein (GFAP) was detected by immunofluorescence.

The platform crossing times of sham operation group, model group and experimental-L, -M, -H groups were (4.58±0.91), (0.73±0.15), (0.92±0.18), (1.58±0.31) and (2.75±0.55), respectively; the error times were (0.50±0.11), (2.08±0.41), (1.75±0.35), (1.58±0.31) and (1.25±0.25), respectively; the relative expression levels of NLRP3 mRNA were 1.00±0.15, 3.51±0.69, 2.94±0.59, 2.57±0.51 and 1.91±0.38, respectively; the relative expression levels of IL-1β mRNA in sham operation group, model group, experimental-H group, control group and oe-NLRP3 group were 1.00±0.17, 2.72±0.54, 1.87±0.37, 1.91±0.38 and 2.66±0.53, respectively; the relative expression levels of IL-10 mRNA were 1.00±0.16, 0.51±0.10, 0.78±0.15, 0.73±0.15 and 0.56±0.11, respectively; the relative expression levels of TNF-α mRNA were 1.00±0.21, 3.08±0.61, 1.77±0.35, 1.82±0.36 and 2.65±0.53, respectively; the relative fluorescence intensities of ROS were 1.00±0.16, 7.22±1.45, 3.05±0.61, 3.13±0.63 and 6.75±1.31, respectively; the relative fluorescence intensities of GFAP were 1.00±0.18, 6.71±1.13, 3.38±0.63, 3.50±0.72 and 5.94±1.19, respectively. The above indexes in the sham operation group were compared with the model group, the above indexes in the experimental-H group were compared with the model group, the above indexes in the oe-NLRP3 group were compared with the control group, and the differences were statistically significant (all P<0.05).

Remimazolam improves postoperative cognitive function in mice by a mechanism that may be related to the inhibition of neuroinflammatory and oxidative stress damage by down-regulating NLRP3 expression.

To observe the effects of different dosage forms of Yupingfeng powder on lipopolysaccharide induced pyroptosis of mouse lung macrophages.

The RAW264.7 cells were randomly divided into normal, model, decoction piece and granule groups. Except for normal group, the other 3 groups were used lipopolysaccharide concentration to conduct a cellular inflammation model. No drug will be given to the normal group, and normal cultivation; the model group was treated with lipopolysaccharide; the decoction piece group was treated with lipopolysaccharide and decoction piece containing serum; the granule group was treated with lipopolysaccharide and granule containing serum. The macrophage inflammatory factor was detected by enzyme-linked immunosorbent assay. The expression levels of cell pyroptosis pathway protein were detected by Western blot.

The tumor necrosis factor -α (TNF-α) contents in cell supernatant of normal, model, decoction piece and granule groups were (48.19±12.00), (154.48±34.84), (125.80±29.66) and (125.73±28.88) pg·mL^-1; the interleukin (IL) -18 levels were (21.36±4.36), (78.33±14.41), (49.77±9.50) and (49.27±9.40) pg·mL^-1; the Caspase-1 levels were (39.72±10.58), (149.49±20.92), (93.25±16.73) and (91.93±16.45) pg·mL^-1; the IL-1β levels were (17.14±3.41), (74.43±16.04), (45.01±9.40) and (43.34±8.96) pg·mL^-1; the relative expression levels of TNF-α mRNA were 1.00±0.05, 2.17±0.07, 1.50±0.20 and 1.35±0.08; the relative expression levels of nuclear factor kappa-B mRNA were 1.00±0.01, 2.02±0.01, 1.52±0.01 and 1.50±0.03; the relative expression levels of IL-18 mRNA were 1.00±0.03, 1.48±0.04, 1.18±0.04 and 1.13±0.10; the relative expression levels of Caspase-1 mRNA were 1.00±0.04, 1.90±0.03, 1.59±0.04 and 1.49±0.10; the relative expression levels of IL-1β mRNA were 1.00±0.10, 1.74±0.08, 1.26±0.06 and 1.23±0.13, respectively. Compared with the model group, there were statistically significant differences of the above indicators in the granule group and the decoction piece group (P<0.05, P<0.01), but there were no statistically significant differences in the above indicators between the decoction piece group and the granule group (all P>0.05).

Both formulations of Yupingfeng powder can reduce the expression level of proteins on the cell pyroptosis pathway and inhibit inflammatory response.

To investigate the mechanism of Banxia-Huanglian (BX-HL) in improving diabetic gastroparesis based on dopaminergic synaptic pathway.

The possible signaling pathways of BX-HL on the treatment of diabetic gastroparesis (DGP) were carried out by network pharmacology methods. The DGP model was constructed by intraperitoneal injection of 60 mg·kg^-1 streptozotocin (STZ) + high-sugar and high-fat diet. The DGP rats were randomly divided into model group and experimental group, with 10 rats in each group; another 10 normal rats were taken as the normal group. The experimental group was administered by gavage with BX-HL solution at a dose of 1.35 g·kg·d^-1, while the normal group and model group were administered by gavage with an equal volume of 0.9% NaCl once a day. The rats in the three groups were administered once a day for 21 days. The gastric emptying rate and intestinal propulsion rate of rats were measured by activated charcoal administration, the levels of neurotransmitter dopamine (DA) in brain tissues were determined by enzyme-linked immunosorbent assay, and the expression levels of protein kinase B (Akt) and glycogen synthase kinase-3 (GSK-3) proteins in brain tissues were determined by Western blotting.

The effective components and corresponding signaling pathways, such as dopaminergic synapses, were screened by various databases for BX-HL on the treatment of diabetic gastroparesis. The fasting blood glucose levels in the normal group, model group and experimental group were (5.61±0.36), (23.40±1.58) and (6.18±0.42) mmol·L^-1; the gastric emptying rates were (71.33±1.23)%, (35.43±3.12)% and (61.59±4.66)%; the intestinal transit rates were (53.19±3.48)%, (29.33±1.91)% and (38.53±2.80)%; the DA contents in brain tissue were (42.43±2.97), (88.20±8.46) and (52.64±5.03) pmol·L^-1; the relative protein expression levels of Akt were 0.67±0.00, 1.35±0.04 and 0.94±0.01; the relative protein expression levels of GSK-3 were 0.96±0.01, 1.24±0.05 and 0.91±0.01, respectively. The above indexes of the experimental group showed statistical significance compared to the model group (all P<0.001).

BX-HL has an ameliorative effect on diabetic gastroparesis in rats, which is mainly reflected in the lowering of blood glucose, the promotion of gastric emptying and intestinal propulsion, the reduction of neurotransmitter DA level, its mechanism may be related to inhibite the Akt/GSK-3 protein expression of dopaminergic synaptic pathway.

To study the effect of etanercept on retinopathy in diabetic rats and explore its mechanism.

Diabetic rat model was established by streptozotocin. The rats were randomly divided into control group (normal feeding), model group (diabetic rat model was established), experimental group (after modeling, etanercept 2 mg·kg^-1 was injected subcutaneously into the abdomen twice a week for 12 weeks), and combined group [based on the experimental group, 100 nmol·mL^-1 microRNA (miR)-30a inhibitor was injected into the tail vein]. The pathological morphology of retina was observed by hematoxylin-eosin staining. Enzyme-linked immunosorbent assay was used to detect the levels of serum inflammatory factors. The positive expression of glial fibrillary acidic protein (GFAP) in retinal of rats was detected by immunofluorescence staining. The relative expression level of miR-30a in retina was detected by reverse transcription real-time fluorescence quantitative polymerase chain reaction.

In the model group, pathological changes occurred in each layer of retina, while in the experimental group, the pathological changes were alleviated, retinal edema was alleviated, cell arrangement was more regular, and the structural boundaries of each layer were clear. In control group, model group and experimental group, the serum levels of interleukin-6 (IL-6) were (0.42±0.07), (1.21±0.16) and (0.69±0.11) μg·L^-1, respectively; the levels of IL-1β were (0.26±0.05), (1.82±0.29) and (0.74±0.16) μg·L^-1, respectively; the levels of tumour necrosis factor-α(TNF-α) were (0.49±0.08), (1.25±0.17) and (0.72±0.13) μg·L^-1, respectively; the relative expression levels of GFAP in retinal tissues were 1.00±0.17, 3.18±0.56 and 1.45±0.23, respectively; the relative expression levels of miR-30a in retinal tissues were 1.00±0.19, 0.61±0.10, and 0.89±0.15, respectively; there were statistical differences between the model group and the control group, and between the experimental group and the model group (all P<0.05).

Etanercept can reduce the level of inflammatory factors in diabetic rats, and has a protective effect on diabetic rats retinopathy, its mechanism may be related to the regulation of miR-30a to activate insulin receptor substrate 1/phosphatidylinositol 3 kinase/protein kinase B signaling pathway.

To explore the protective mechanism of Celosin Ⅰ on retinal ganglion cells by regulating reactive oxygen species (ROS) mediated mitochondrial apoptosis pathway.

Twenty-four New Zealand white rabbits were randomly divided into sham-operation, model, control and experimental groups, with 6 rabbits per group. The sham-operation group only had the bulbar conjunctiva cut and sutured, the other 3 groups were received with optic nerve clamp injury to contruct the optic nerve injury model. The sham-operation and model groups were given equal volumes of physiological saline by gavage, the control group was given a dose of 0.15 mg·kg^-1 mecobalamine solution by gavage, and the experimental group was given a dose of 30 mg·kg^-1 Celosin Ⅰ solution by gavage. Four groups were administered a daily gavage treatment for four consecutive weeks. The Tunnel method was used to detect apoptosis of retinal ganglion cells, and immunofluorescence was employed to detect the expression of ROS, superoxide dismutase (SOD) and caspase in retinal tissues. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-like protein 1 (Bcl-xl), Bcl-2-associated X protein (Bax), and cytochrome C (CytC) proteins in retinal tissues were assessed using Western blotting.

After four weeks of intervention, the apoptosis rates of ganglion cells in the experimental, control, model and sham-operated groups were (7.65±1.20)%, (11.65±1.29)%, (17.66±0.93)% and (0.97±0.14)%; the expression levels of ROS in retinal tissues were 6.14±0.57, 8.25±0.96, 16.51±1.07 and 2.05±0.23; the expression levels of SOD were 2.45±0.33, 1.48±0.23, 0.74±0.13 and 3.19±0.30; the expression levels of caspase-3 were 8.55±1.11, 12.68±0.92, 18.74±1.97 and 3.08±0.20; the expression levels of caspase-9 were 3.69±1.20, 7.79±1.13, 12.81±1.72 and 2.31±0.30; the relative expression levels of Bcl-2 were 0.46±0.02, 0.64±0.07, 0.77±0.05 and 0.20±0.03; the relative expression levels of Bcl-xl were 0.46±0.05, 0.61±0.05, 0.78±0.03, and 0.20±0.03; the relative expression levels of Bax were 0.50±0.07, 0.69±0.18, 0.82±0.13 and 0.14±0.04; the relative expression levels of CytC were 0.43±0.15, 0.80±0.14, 1.08±0.19 and 0.26±0.07. The differences in the aforementioned indicators in the experimental group compared with control group were statistically significant (all P<0.05).

Celosin Ⅰ reduces ROS levels in retinal tissue of a model of optic nerve injury, inhibits the expression of mitochondrial apoptosis related proteins in retinal ganglion cells, and reduces retinal ganglion cell apoptosis.

To investigate the role of the receptor-interacting protein kinase 3 (RIP3)-mediated necroptosis pathway in penile tissue damage in diabetes mellitus-induced erectile dysfunction (DMED) rats and the effects of GSK872 and Yimusake intervention in DMED rats.

Male SD rats with normal sexual function were selected, and diabetic rats were constructed by 2 consecutive days injections of streptozotocin (45 mg·kg^-1), DMED rats were screened and randomly divided into model group, experimental group, Yimusake group and combined group. The normal and model groups were injected intraperitoneally with an equal amount of 0.9% NaCl and gavaged with an equal amount of distilled water; the experimental group was injected intraperitoneally with 1 mg·kg^-1 GSK872 and gavaged with an equal amount of distilled water; the Yimusake group was injected intraperitoneally with an equal amount of 0.9% NaCl and gavaged with 250 mg·kg^-1 Yimusake; the combined group was injected intraperitoneally with 1 mg·kg^-1 GSK872 and gavaged with 250 mg·kg^-1 Yimusake. The expression of RIP3, mixed lineage kinase domain like protein (MLKL) and transient receptor potential melastatin 7 (TRPM7) were detected by immunohistochemistry and immunofluorescence, α-smooth muscle actin (α-SMA) and Collagen Ⅰ were detected by Western blotting in rat penile tissues.

The positive area ratios of RIP3 in the penile tissues of normal, model, experimental, Yimusake and combined groups were (7.71±1.92)%, (35.72±2.73)%, (20.20±2.51)%, (19.94±2.66)% and (9.31±1.98)%; MLKL positive area ratios were (7.39±1.73)%, (26.48±1.74)%, (15.66±1.87)%, (15.88±1.59)% and (8.45±1.65)%; TRPM7 positive area ratios were (6.43±2.10)%, (35.77±1.78)%, (23.76±1.79)%, (25.24±2.08)% and (9.39±1.72)%; α-SMA protein relative expression levels were 1.53±0.13, 0.39±0.08, 0.88±0.14, 0.86±0.12 and 1.52±0.12; Collagen Ⅰ protein relative expression levels were 0.29±0.10, 1.20±0.14, 0.73±0.07, 0.71±0.11 and 0.31±0.10. The differences between the above indices in the model group compared with the normal group, the above indices in the experimental group compared with the model group, the above indices in the Yimusake group compared with the model group and the above indices in the combined group compared with the model group were all statistically significant (all P<0.05).

RIP3/MLKL/TRPM7 necroptosis pathway may participate in regulating the progression of DMED rats by promoting cell death and fibrosis, while GSK872 and Yimusake may ameliorate penile injury in rats by inhibiting this pathway.

To explore the effects and mechanism of zoledronic acid on bone metabolism in osteoporosis rats under hypoxic environment.

The rat model of osteoporosis in hypoxia environment was established by bilateral ovary extraction combined with low-pressure oxygen chamber feeding, and was randomly divided into model group, control group and experimental -L, -M, -H groups, with 10 rats per group; another 10 normal rats were selected as the normal group. Experimental -L, -M, -H groups were given 2.5, 5.0 and 10.0 mg·kg^-1 zoledronic acid by subcutaneous injection, once a week. Control group was given 0.105 mg·kg^-1 estradiol valerate by intragastric administration, once a day. Normal and model groups were injected with equivalent 0.9% NaCl by subcutaneous injection. Six groups were treated for 4 weeks. The serum levels of tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and bone gla protein (BGP) were detected by enzyme-related immunosorbent assay. The bone mineral density (BMD) was measured by dual-energy X-ray. The expression levels of mitogen extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) in femur were detected by Western blot.

The TRAP levels of experimental-M, experimental -H, control, model and normal groups were (78.06±1.16), (69.39±1.44), (70.19±1.24), (94.39±1.04) and (64.95±1.19) U·L^-1; the ALP levels were (61.72±3.10), (45.40±0.82), (43.53±1.52), (88.69±2.29) and (42.01±1.56) U·L^-1; the BGP levels were (14.82±0.28), (12.29±0.28), (12.92±0.29), (19.96±0.46) and (11.79±0.59) ng·mL^-1; the BMD levels were (0.28±0.02), (0.35±0.04), (0.38±0.03), (0.16±0.02) and (0.41±0.06) g·cm^-2; the relative expression levels of p-MEK protein were 0.64±0.06, 0.84±0.05, 0.78±0.01, 0.15±0.06 and 0.88±0.04; the relative expression levels of p-ERK protein were 0.70±0.03, 0.95±0.02, 0.84±0.07, 0.29±0.03 and 0.97±0.06, respectively. Compared with the model group, the above indexes in the experimental-M, -H groups and control group were statistically significant (all P<0.05).

Zoledronic acid can improve bone mineral density and bone metabolism in hypoxic osteoporosis rats, the mechanism may be related to the regulation of MEK/ERK pathway.

To establish a method for the quantification of aripiprazole in male SD rats muscle, and apply it to the research of drug residue at injection site in rats after administration of long-acting aripiprazole injection (LAI), and to compare the pharmacodynamics in mice.

An ultra-high performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) assay was used to determine the concentration of aripiprazole in rats muscle. Sample pre-treatment: Muscle tissue was homogenized in a 30% acetonitrile solution containing 5% acetic acid, and then precipitated with 0.1% formic acid in acetonitrile for detection. Chromatography column: Waters HSS T3^TM C₁₈ column (2.1 mm×50.0 mm, 1.8 μm). Mobile phase: 5% acetonitrile and 95% acetonitrile both containing 0.1% formic acid. Flow rate: 0.6 mL·min^-1. Injection volume: 5 μL. Mass spectrometry conditions: electrospray ion-source positive ions mode, multiple reaction monitor (MRM) scanning mode. After intramuscular administration of 25 mg·mL^-1 aripiprazole microspheres for injection (MS) or long-acting aripiprazole for injection (brand name: Abilify Maintena^®, AM), the drug residue in male SD rats was determined using an UPLC-MS/MS method. The hyperlocomotion model was established by acute administration of phencyclidine (PCP). And a comparative study was conducted on the efficacy of MS (40, 80, 240 mg·kg^-1) and AM (80 mg·kg^-1).

After 28 days of administration, the drug residue ratio of MS group was 6.27%-12.38%, while that of AM group was 23.31%. After 35 days of administration, the average drug residue ratios were 0.58%-2.94% and 10.28%-18.87%, respectively. Both the AM group and each dose (40, 80, 240 mg·kg^-1) of MS group could inhibit the spontaneous movement of mice induced by PCP.

The inhibitory effect of Aripiprazole Microsphere for injection is comparable to that of Abilify Maintena on the spontaneous movement of mice caused by PCP. And there is a certain dose-response relationship for MS, while the drug residue at the injection site of MS is much lower than that of Abilify Maintena.

To establish in vitro and in vivo correlations from in vitro dissolution data and in vivo data from animals.

In vitro method: an aqueous gelatin solution was first prepared with alkaline gelatin, paliperidone palmitate was added into the solution and mixed well, gelatin hydrogel was obtained by refrigerating in the refrigerator for 1-2 h. The hydrogel was put into paddle dissolution apparatus for dissolution experiments, the concentration of the drug was detected by high-performance liquid chromatography and the degree of release was calculated. In vivo method: Timing was started after paliperidone palmitate was injected into Beagle dogs via intramuscular injection, blood was collected at 0 h before and 6 h-36 d after drug administration, respectively, the blood concentration was detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique and the blood concentration-time curve was plotted. Finally, the obtained in vitro data were fitted by Weibull function, the in vivo data were transformed by Nelson-Wagner method and area under the curve method. Graphs were constructed to evaluate the correlation between in vivo and in vitro drug release.

The in vitro release was approaching 100% by day 13, while the in vivo release was approaching 100% by day 30-36. Both Nelson-Wagner method and area under the curve method yielded that in vivo and in vitro were correlated.

It is feasible to establish in vitro correlations from in vivo dissolution data and in vivo data from animals.

To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of crizotinib in human plasma and apply this method to determine its concentration in patients with non-small cell lung cancer after administrating.

Osimertinib was employed as the internal standard. The plasma samples were performed through protein precipitation with acetonitrile and chromatographed on Agilent Eclipse XDB C₁₈ (2.1 mm×100.0 mm, 3.5 μm), mobile phase was consisted of acetonitrile containing 0.1% formic acid - 10 mmol·L^-1 ammonium acetate containing 0.1% formic acid, flow rate was 0.25 mL·min^-1, the column temperature was 30 ℃. The whole analytical time was 3 min. The detection was performed by positive ion electrospray ionization in multiple reaction monitoring mode, the quantitative analysis ion pairs were m/z 450.1→259.2 and m/z 500.2→72.1. The specificity, standard curve and lower limit of quantitation, precision and recovery, stability, matrix effect and residual effect of the method were investigated. After full validation, the method was applied to the determination of crizotinib in human plasma.

Good linearity was observed over the concentration range of 10.0-2 500.0 μg·L^-1, the calibration curve was y=4.54×10^-3x+3.22×10^-2 (r=0.992 7). The accuracies were between 97.66%-105.55%. The relative standard deviation of inter-day and intra-day precision were less than 8.49%. The average recoveries and matrix effects were between 80.10%- 86.05% and 95.22%-110.66%.

The LC-MS/MS method is developed which meets the requirements of biological sample analysis and it can quickly, accurately monitor the concentration of crizotinib.