Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome

Objective To investigate the effect and mechanism of linarin(LA) in an experimental dry eye model.Methods LA or vehicle was applied in two dry eye models: an in vitro hyperosmotic stress model and an in vivo desiccating stress(DS) murine model. The viability of human corneal epithelial cells(HCECs) was measured using a cell counting kit(CCK-8).Tear secretion was assessed using the phenol red cotton test. The tear break-up time(TBUT) was recorded using 0.1% liquid fluorescein sodium. Corneal epithelial permeability was evaluated through Oregon green dextran(OGD) staining.Conjunctival goblet cells were counted using periodic acid-Schiff(PAS) staining. Terminal deoxynucleotidyl transfer d UTP nickend labeling(TUNEL) staining was used to quantify apoptotic cells in both models. The expression of Ki-67 was measured in HCECs in the cell model while that of matrix metalloproteinase(MMP)-3 and-9 was measured in the murine model through immunofluorescence staining. Real-time quantitative PCR(RTqPCR) was performed to assess the expression of MMP-3 and MMP-9 in the corneal epithelium and NLRP3, ASC, Caspase-1,interleukin(IL)-1β, IL-18, and tumor necrosis factor(TNF)-α in the conjunctiva. The protein expression levels of NLRP3, ASC,Caspase-1, IL-1β, and IL-18 in the conjunctiva were assessed via Western blot.Results In the in vitro model, treatment of HCECs with LA showed no toxicity, increased proliferation, and reduced apoptosis. In the murine model, compared to the control, LA significantly increased tear production and TBUT, improved OGD staining, and increased the number of goblet cells. Topical treatment of LA to mice provided decreased expression of MMP-3, MMP-9, TNF-α, and apoptotic corneal epithelium. Topical administration of LA also suppressed the NLRP3 inflammasome in the dry eye disease(DED) murine model by decreasing the expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the conjunctiva.Conclusion Our findings support the safety and efficacy of LA in the treatment of DED. LA alleviated corneal epithelial damage and suppressed NLRP3 inflammasome-mediated immunity in the conjunctiva in a murine model of DED.

Inflammatory Mechanism of Total Flavonoids of Chrysanthemum and Medicated Serum on Castrated Dry Eye Animal and Cell Models

Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.

Effect of Flos Buddlejae Granules on Apoptosis Factors Bax,Bcl-2,Fas and FasL in Lacrimal Gland Cells of Castrated Male Rabbits

Objective To observe the effect of different concentration Flos Buddlejae(Mi Meng Hua,密蒙花)granules on apoptosis factors of lacrimal gland cells of castrated male rabbits,and study the treatment effects of Flos Buddlejae(Mi Meng Hua,密蒙花)granules in the dry eye model of castrated male rabbits.Methods Flos Buddlejae(Mi Meng Hua,密蒙花)raw material was made into granules.Thirty healthy adult New Zealand rabbits were randomly divided into five groups,six rabbits in each group.Group A:blank group,Group B:model group,Group C:Flos Buddlejae(Mi Meng Hua,密蒙花)granule group,Group D:placebo group,Group E:testosterone group.Except for group A,all rabbits underwent removal of bilateral testis and epididymis.Rabbits in group C were administered Flos Buddlejae(Mi Meng Hua,密蒙花)granules(100 mg/kg),three times per day;rabbits in group D were administered normal saline,three times per day.Rabbits in group E were injected with testosterone propionate(0.5 mL/kg)in the thigh muscle,every 3 days.All rabbits were tested by Schirmer I test(SIT)and tear film break-up time(BUT)before operation and 4 weeks after operation.After 4 weeks,all rabbits were sacrificed by air embolism and clipping of the lacrimal gland.Apoptosis factors,including Bax,Bcl-2,Fas and FasL of lacrimal gland cells were characterized by immunohistochemistry,and resulting data were statistically analyzed.Results(1)Comparison of SIT and BUT before and after operation:There were statistically significant differences between groups B and D(P<0.01),but not among other groups(P>0.05).(2)Comparison of apoptosis factors Bax,Bcl-2 Fas and FasL:In a comparison of groups B and D,there was no statistically significant difference after operation(P>0.05).In a comparison of the other groups,there were statistically significant differences(P<0.01).In comparisons among A,C and E groups,there were no statistically significant differences(P>0.05).Conclusion Compared with androgen,Flos Buddlejae(Mi Meng Hua,密蒙花)granules caused similar but slightly weaker depression of Bax,Fas and FasL,and increased expression of Bcl-2.