Effects Of Mingmu Xiaoyao Granule On Retinal Function In Rat Model Of Chronic Unpredictable Mild Stres

Clinically,we have found that many patients with chronic retinal diseases are accompanied by anxiety and depression.And this state of anxiety and depression,in traditional Chinese medicine belongs to liver depression syndrome.Old Chinese medicine doctor Pang Zanxiang emphasized the theory of "eye diseases with multiple depression" and pointed out that the good treatment of eye diseases must first relieve depression.Therefore,we discussed the connection between liver and objective physiological and pathological in Review 1.Chronic unpredictable mild stress(CUMS)is a classic stimulus method to induce anxiety and depression in rats,often causing autophagy and oxidative stress.Therefore,the effects of CUMS stimulation on autophagy and oxidative stress were discussed in Review 2.However,the effect of CUMS stimulation on retina has not been reported.Thus we designed animal experiments.Objective:To study the effect of Mingmu Xiaoyao granule(MMXY)on retinal morphology and function of rats with CUMS-induced anxiety and depression and its pathogenesis.Methods:Animal model and grouping:52 male SD rats were randomly divided into control group(n=14)and CUMS group(n=38).At 4 weeks,6 animals were randomly selected from each group,and the retina was isolated and histologically examined.The results indicated that the modeling was successful.At the 5th week,CUMS group rats were randomly divided into 4 groups with 8 rats in each group.Model group(CUMS+pure water),MMXY-H(CUMS+MMXY 7.2 g/kg/d),MMXY-L(CUMS+MMXY 3.6 g/kg/d)and Carbamazepine group(CUMS+CBZ 20 mg/kg/d),all rats were given corresponding intragastric drug,once a day.At 12 weeks,sucrose preference and open field tests(SPT and OFT)were performed to evaluate the anxiety and depression status of rats.In live rats,optical coherence tomography angiography(OCTA)was used to measure retinal thickness and blood flow,while electroretinograms(ERGs)and visual evoked potentials(VEPs)were used to evaluate retinal function.The next day,the specimens were sacrificed for serological,histological,immunofluorescence,Western blot(WB)and transmission electron microscopy examinations to explore the mechanism of MMXY in CUMS rats.Results:We elaborated from the following four aspects,the main results are as follows:The first part is the establishment and evaluation of CUMS-induced anxious-depressive rat model.1.MMXY is a good prescription for the treatment of retinal diseases of liver depression and qi stagnation.As the observation drug,carbamazepine was selected as the positive control drug.CUMS stimulation is a classic model of induced anxiety-depression in rats,which is a good model for simulating persistent anxiety and depression in humans.The sucrose preference and open field test were used to evaluate behavioral changes in rats.2.CUMS-induced anxious-depression-like rat model was successfully established at 4 weeks.The rats showed behavioral abnormalities and mild retinal conduction abnormalities.In the second part,MMXY improved the anxiety-depression-like behavior and reversed the imbalance of serum hormones and neurotransmitters in CUMS rats.1.In terms of behavior,the decrease of body weight,sucrose preference and open field activity at weeks 8 and 12 reflected that the rats had been in a state of anxiety and depression.However,MMXY-H group significantly improved the above abnormalities in CUMS rats,suggesting that MMXY improved the anxiety-depression-like behavior in rats.2.At 12 weeks,dopamine(DA)in the Model group was significantly decreased and corticosterone(CORT)was significantly increased,reflecting the decreased excitability of rats.Compared with Model group,both DA and CORT in MMXY-H group were improved,suggesting that MMXY reversed the imbalance of serum hormones and neurotransmitters.In the third part,MMXY improved retinal structure and function in CUMS rats.1.In terms of retinal morphology in rats:In Model group,ultrastructural damage was observed under electron microscope,including nuclear contraction of retinal ganglion cells(RGC),destruction of organelles such as mitochondria,and swelling of synapses and mitochondria in inner plexus layer(IPL).Under light microscope,RGC showed nuclear pyretosis and decreased cytoplasm,IPL was obviously thinner,and the number and volume of cells in inner nuclear layer(INL)and outer nuclear layer(ONL)were reduced.Compared with the control group,the morphology of retinal layers were significantly damaged under electron microscope and light microscope,suggesting that CUMS induced the destruction of retinal morphology and structure.However,MMXY significantly improved the ultrastructure and optical structure of CUMS rats.2.In terms of retinal thickness,the thinning of retinal thickness of Model group rats was confirmed by HE staining and OCTA measurement.MMXY improved retinal thickness in CUMS rats.3.In terms of retinal blood flow,OCTA measurement and OS2 wave amplitude measurement of retinal electrogram confirmed the decrease of inner retinal blood flow of Model group rats.MMXY improved blood flow in the inner retina.In terms of systemic hemagglutination indexes of rats,Model group rats showed hypercoagulability,which was improved by MMXY.These results indicated that MMXY improved blood flow in the inner retina of rats.4.OCTA was used to measure the thickness and blood flow of each retinal layer and choroid of rats,and to improve the correlation analysis between thickness and blood flow.The results showed that CUMS stimulation had the greatest effect on the inner retinal layer of rats,and MMXY could improve the thickness by preferentially improving the blood flow of retinal nerve fiber layer(RNFL).5.ERGs and VEPs were used to evaluate retinal function.The results showed that at 12 weeks,the photoreceptor cells,bipolar cells and RGC cell tertiary neurons and synapses were impaired,as well as the optic nerve conduction dysfunction.However,MMXY significantly improved the amplitude and peak time of the above waveforms,combined with the improvement of histological morphology under light and electron microscopy,and finally showed the improvement of rat optic cells and visual conduction function.In the fourth part,the mechanism of improving retinal function in CUMS rats with MMXY was discussed.In the study of the mechanism of CUMS causing retinal abnormalities in rats,we found two pathways,autophagy and oxidative stress.It is described as follows:Mechanism 1:MMXY protects retinal function in CUMS rats by inhibiting autophagy.1.Electron microscope results showed obvious autophagy bodies in the Model group,indicating autophagy occurred in the Model group.Moreover,the high expressions of microtubule-associated proteins light chain 3(LC3)and Beclinl in the Model group by immunofluorescence and WB indicated a high autophagy flux in the Model group.However,MMXY decreased the expression of LC3 and Beclinl,and decreased the autophagy flux.The above results showed that high flux autophagy occurred in the Model group,causing damage to retinal structure.2.Mechanism of retinal autophagy:(1)PI3K/Akt/mTOR signaling pathway is a classic autophagy regulatory pathway.In our study,the ratios of the four phosphorylated proteins P-PI3K p85,p-Akt(Ser473),p-Akt(Thr308)and p-mTCR(Ser2448)to the corresponding total proteins in the Model group were significantly reduced compared with those in the Control group(p<0.01).Moreover,the changes of the three phosphorylation proteins in the PI3K/Akt/mTOR signaling pathway were consistent,suggesting that the activity of PI3K/Akt/mTOR signaling pathway was low in the Model group,autophagy inhibition was reduced,autophagy was enhanced,and retinal tissue was destroyed.Increased intraocular pressure in Model group also increased retinal autophagy.3.MMXY inhibits retinal autophagy:The above four phosphorylated proteins were significantly increased in MMXY-H group,suggesting that MMXY promoted the expression of PI3K/Akt/mTOR signaling pathway related proteins,reduced the autophagy flux,reduced the damage of retinal morphology and function,improved the damage of synapses between neurons,and reduced intraocular pressure.In many ways,it reduced the damage of autophagy on retina.It reverses the structure and function of the retina.Mechanism 2:MMXY protects retinal function in CUMS rats by inhibiting oxidative stress.1.CUMS stimulation induced the enhancement of oxidative stress in rats.In the Model group,increased malondialdehyde(MDA),decreased superoxide dismutase(SOD)in serum,and tumor necrosis factor alpha(TNF-α)and interleukin-12B(IL-12B)inflammatory factors were elevated,suggesting oxidative stress.Furthermore,mitochondrial swelling and fracture were observed in the multilayer retinal structures of the model group,suggesting mitochondrial dysfunction.And low expression of heme oxygenase-1(HO-1)oxidative stress protein in retina by WB detection.In summary,it is concluded that obvious oxidative stress occurred in the retina of Model group.2.Oxidative stress occurred in the retina of CUMS rats,which caused damage to various layers of tissues,including damage to photoreceptor cells,RGC,retinal pigment epithelium(RPE),decrease in the number of synapses and impairment of optic nerve conduction function.3.MMXY inhibits oxidative stress in CUMS rats:MMXY improves the morphology of mitochondria in the multilayer structure of retina,reduces the damage of photoreceptor cells,RGC,PRE layer caused by oxidative stress,increases the number of synapses between neurons,promotes the improvement of optic nerve conduction function,and finally protects retinal function.4.As for the relationship between retinal autophagy and oxidative stress in CUMS rats,our results showed that the autophagy flux and oxidative stress level in the Model group were significantly increased compared with that in the control group,suggesting that oxidative stress may promote the enhancement of autophagy or autophagy is enhanced in response to oxidative stress,and the two effects have the same trend,eventually leading to retinal tissue damage.In MMXY group,the expression of PI3K/Akt/mTOR pathway was enhanced,and the autophagy flux and oxidative stress levels were also decreased,which finally had a protective effect on retinal tissues.Conclusions:MMXY can inhibit autophagy and reduce oxidative stress damage by regulating PI3K/Akt/mTOR pathway,improve anxiety and depression-like behavior in CUMS rats,and reverse retinal morphological and functional abnormalities.