Effects of Shumu Decoction on Pathological Changes of Cornea in Patients with Diabetic Dry Eye

BACKGROUND:To observe the pathological changes of cornea and to clarify the effect Shumu Decoction on the cornea in diabetic dry eye. METHODS:The study assessed 200 eyes of 100 patients with diabetic dry eye.The treatment group 50 patients(100 eyes) and the control group 50 patients(100 eyes). The control group was treated with sodium hyaluronate eye drops. On the basis of the control group, the treatment group was treated by oral administration of Shumu Decoction.All participants underwent the examination of laser confocal microscope to obtain the image of cornea which were used to evaluate the differences between the two groups before and after treatment. RESULTS:After treatment,The corneal epithelial cell density in the treatment group was signicantly higher than that in the control group(P <0.05) .The cell area of the two groups increased compared with that before treatment, but the area in the control group was signicantly larger than that in the treatment group (P<0.05).The number of Langerhans cells in the control group were signicantly higher than that in the treatment group(P<0.05).The morphology of Langerhans cells was characterized by large area and pseudopodia in the control group.The density, length of central corneal nerve bers in the two groups were reduced compared to before treatment, but the decrease of nerve ber in the control group was more obvious than that in the treatment group(P<0.05), and the morphology of nerve bers was obvious change in the control group(P<0.05).At 4 weeks' treatment,the corneal endothelial cells showed morphological abnormality, there was signicant difference in the ratio of endothelial cell hexagon between the two groups, and the corneal thickness increased signicantly in the control group(P<0.05). CONCLUSION:Shumu Decoction can delay the loss of corneal cells and nerve bers restore the normal function of cornea.Those may

morphological abnormality, there was signi cant difference in the ratio of endothelial cell hexagon between the two groups, and the corneal thickness increased signi cantly in the control group(P<0.05).
CONCLUSION:Shumu Decoction can delay the loss of corneal cells and nerve bers and restore the normal function of cornea.Those changes may alleviate the symptoms of patients with diabetic dry eye Background Diabetic dry eye is one of diabetic ocular surface disorders. Epidemiological study has found that about 50% of diabetes concomitant dry eye disease [1]. Various pathological changes of cornea caused by diabetes are the main causes of dry eye disease [2]. Persistent corneal epithelial defects cause many symptoms of dry eye disease, such as dryness, photophobia, foreign body sensation and so on.Decreased corneal sensitivity affect the production of re exive tears and the stability of tear lm.In addition, The amount of tear was signi cantly reduced in patients with diabetic dry eyes, but the patients had no obvious clinical feature of dry eye disease,which might be related to the changes in corneal structure and function caused by diabetes [3][4], indicating that the corneal abnormality closely associated with occurrence of diabetic dry eye.
Qi de ciency and yin de ciency are the main syndrome grouping of Traditional Chinese Medicine (TCM) of diabetic dry eye. Now treatment is mainly based on the method of nourishing qi and nourishing yin.
Although this treatment could improve the clinical symptoms of diabetic dry eye,it was not clear how did TCM play a role and how did TCM affect cornea.In this paper, the changes of cells and nerve bers in cornea were observed by laser confocal microscopy and then the effects of Yiqi Yangyin Chinese medicine on the cornea were clari ed.

Ethical Approval
The study protocol was approved by the Ethics Committee of Shaanxi traditional Chinese medicine hospital and adhered to the tenets of the Declaration of Helsinki. Before the examination, we have got written informed consents from all subjects.

Subjects
In this study, 200 eyes of 100 patients with diabetic dry eye patients (56 males, 44 females; mean age 51.9 ± 9.5y) were evaluated in Shaanxi traditional Chinese medicine hospital during the period from January 2018 to December 2019. The mean duration of diabetes was 7.7±3.41y (range, 5-20y). The patients were those who meet the diagnostic criteria of type 2 diabetes and dry eye disease,and those who meet the diagnostic criteria of syndrome grouping of TCM of dry eye disease with de ciency of both qi and Yin,and those who have not used local drugs for eyes within one month before entering the group.Exclusion criteria included type 1 diabetes and monocular disease,allergy to research drugs, pregnant or lactating women,ocular and systemic diseases(conjunctivitis,keratitis, iris, lacrimal duct and other local eye diseases,SjÖgren's syndrome and rheumatoid arthritis).

Research methods
All participants were randomly divided into two groups: the treatment group 50 patients(100 eyes) and the control group 50 patients(100 eyes). The control group was treated with sodium hyaluronate eye drops: three times a day, one drop each time, for four weeks as a cycle of treatment. On the basis of the control group, the treatment group was treated by oral administration of Shumu Decoction(Yiqi Yangyin Chinese medicine).The prescription was:(Astragalus membranaceus 15g,Rehmannia glutinosa Libosch 12g,Dendrobium candidum 12g, Polygonatum sibiricum 15g, Paeonia lacti ora Pall 10g, Lycopus lucidus 12g, Astragali Complanati Semen 12g,Cuscuta chinensis Lam 12g, Cyperus rotundus Linn 9g). The raw medicine was provided by Shaanxi traditional Chinese medicine hospital.Taking method:the decoction was processed into a vacuum package by the Chinese pharmacy of the hospital. Each bag was 200ml, one bag a time,twice a day.
Before and 4 weeks after the treatment, the cornea of all participants were examined by laser confocal microscopy.
Laser Confocal Microscope Examination A ll participants were examined by laser confocal microscope (Heidelberg company, Germany).The eye of participants were topically anesthetized with articaine eye drops (Alcon company, USA) for three times. The operator rstly droped Visdisc eye gel (Bausch & Lomb company, USA) on the surface of laser confocal microscope lens and then covered sterile corneal contact cap on microscope lens. The mandible and forehead of the participants were xed on the Laser confocal microscope respectively. The participants were asked to look at the xation lamp, The operator adjusted the position of the objective lens to make the contact cap contact with the central cornea slightly.The operator rotated the focal plane adjusting ring to obtain the image of each layer of cornea, and collected and saved the image. The number of nerve bers was the total number of nerve bers in one image. At least three images were analyzed and the average value was taken.

Statistical Analysis
The measurement data was expressed by mean ± standard deviation (), and analyzed by SPSS 22.0 statistical software. Before and after treatment, paired sample t test was used, and two independent samples t test was used. The difference was statistically signi cant (P < 0.05).

Corneal epithelial cells and Langerhans cells
The corneal epithelial cell density in both groups decreased after treatment, but the cell density in the treatment group was signi cantly higher than that in the control group, with a signi cant difference (P <0.05) ( Table 1).After treatment, the cell area of the two groups increased compared with that before treatment, but the area in the control group was signi cantly larger than that in the treatment group, and the difference was statistically signi cant (P<0.05) ( Table 1). Table 1 Comparison of cell density (cells / mm -2 ) and surface cell area (µm -2 ) of corneal epithelium in two groups After treatment,the number of Langerhans cells in the treatment group was not signi cantly increased, while the number of cells was signi cantly increased in the control group,the difference between the two groups was statistically signi cant (P<0.05).At the same time,The morphology of Langerhans cells were characterized by large area and pseudopodia (Table 2, Figure 1).

Nerve bers
After treatment, the density, length, and branch density of central corneal nerve bers in the two groups were reduced compared to before treatment, but the density and length of nerve bers in the treatment group were signi cantly higher than those in the control group, and the difference between the two groups was statistically signi cant.(P <0.05).
After treatment, The morphology of nerve bers was not obvious change in the treatment group, while nerve ber curvature in the control group increased signi cantly, and the difference between the two groups was statistically signi cant (Table 3).

Corneal stroma
The corneal stroma is divided into ve layers from the back to the front. After treatment, the cell density was(946±21/ mm 2 and 1074±12 / mm 2 in 0% to 10% depth;849±12 / mm 2 and 939 ± 9 / mm 2 in 11%-33% depth)in two groups and the cell density was decreased signi cantly in the control group.The difference between the two groups was statistically signi cant (P <0.05).
There was no signi cant difference in the cell density of deep cornea between the two groups.

Corneal endothelial cells and corneal thickness
After treatment, corneal endothelial cells in the control group showed morphological changes, the cells lost normal regular hexagonal morphology and showed irregular morphology (cell polymorphism) ( Table  4) (Figure 1),There was signi cant difference in the ratio of endothelial cell hexagon between the two groups.In addition, there was no signi cant change in the density of endothelial cells between the two groups.
After treatment, the corneal thickness increased signi cantly in the control group(564µm and 520µm,respectively), and the difference was statistically signi cant (P <0.05).

Discussion
At present, the treatment focused on improving clinical undesirable symptoms of dry eye disease rather than curing.Chinese medicine treatment had obvious advantages in restoring eye surface health, improving eye discomfort, protecting visual function, reducing the recurrence of dry eye disease.In our long-term clinical practice, combined with the clinical characteristics of diabetic dry eye and the experience of dialectical treatment, It was believed that the etiology and pathogenesis of diabetic dry eye, though varied and complicated, were always based on Qi de ciency and yin de ciency, with dry heat as its target.According to this understanding, we proposed the method of bene ting qi and nourishing Yin as the treatment principle for diabetic dry eye, and apply the herbs of nourishing qi and Yin such as Astragalus membranaceus, Rehmannia glutinosa Libosch, Dendrobium candidum,and Polygonatum sibiricum to the treatment of diabetic dry eye. In addition, Zhang analyzed the etiology and pathogenesis of diabetic dry eye, and thought that "de ciency of both Qi and Yin, stagnation of eye and meridians" were the main pathogenesis of diabetic dry eye [5].
In the condition of high blood glucose, the tactile threshold of cornea increased, the shape of nerve ber became thin and the number of nerve ber became decreased, the sensitivity of cornea and immune defense of ocular surface were decreased, corneal infection and corneal ulcer appeared, which eventually led to the disorder of ocular surface and induced the morbidity of dry eye disease [6].In addition, the decrease of corneal sensation caused by hyperglycemia reduced tear production. The decrease of corneal innervation was related to the abnormal tear lm function and closely related to the severity of dry eye disease [7]. Therefore,cornea was the main tissue invaded by diabetic dry eye, and the clinical symptoms of dry eye disease could be further aggravated after keratopathy, forming a vicious circle between the two.
Diabetes led to a series of pathological changes in corneal epithelial cells,such as the cellular pleomorphism, irregular arrangement and reducing proliferation and decreasing cell density [8]. Our study found that after the treatment the number of epithelial cells mildly decreased compared with the control group, and the thickness of cornea was not obvious increased. These changes indicated that Shumu Decoction could not only improve the effect of hyperglycemia on the morphology and number of corneal epithelial cells, but also enhance the function of corneal epithelial barrier, signi cantly reduce corneal edema. Previous research found that the use of Yiqi Runmu Chinese medicine could protect cornea and conjunctiva from damage and restore the function of ocular surface barrier in hypertonic saline-induced dry eye mouse models,which was consistent with our ndings [9].
In addition, we found that the density of Langerhans cells was signi cantly increased in the control group. The reason was that the persistent high blood glucose levels made the corneal tissue in the state of hypoxia, causing chronic in ammation of the cornea, and then stimulating the Langerhans cells as antigen presenting cells, and the Langerhans cells had the function of ingesting, processing and presenting antigens, which could cause a variety of in ammatory cells to participate in the ocular surface immune-in ammatory response, resulting in an increase in the density of Langerhans cells [10], while the number of Langerhans cells in the treatment group only slightly increased, indicating that Chinese medicine could improve the symptoms of dry eye increase caused by diabetes by inhibiting the in ammatory response in the cornea,restoring the normal function of cornea.It was found in the rats model of diabetic dry eye that Yiqi Shengjin Chinese medicine (Fuming Tablet) reduce the secretion of IL-6, TNF-a and IL-1β in the tears of rats and the expression of TGF-β1 in the cornea epithelium cell, thereby reducing the level of in ammatory factors on the ocular surface and improving the discomfort symptoms of eye [11].In accordance with our results, it was con rmed that Yiqi Yangyin Chinese medicine can restore the immune barrier of cornea by down-regulating the in ammatory level in cornea so as to achieve the purpose of curing diabetic dry eye.
Recent studies had shown that the density of corneal nerve ber decreases in people with diabetes and progresses gradually with the duration of diabetes [12]. In this study, we found that in the control group, the number and the length of the nerve bers were signi cantly reduced. In addition, the morphology was signi cantly changed, and the curvature was increased. The number, length and the shape of nerve bers were signi cantly improved after oral administration of Shumu Decoction. It was speculated that damage of corneal epithelial cell integrity lead to morphological changes of nerve bers which eventually lead to the decrease of the number of nerve bers. After taking Chinese medicine, corneal integrity could be signi cantly restored, and the nerve bers could be protected from the effects of hyperglycemia.Studies have con rmed that Yangyinshengjin Chinese medicine (Dendrobium o cinale) could inhibit or correct abnormal changes in the activity of polyhydric alcohol dehydrogenase and aldose reductase in cells stimulated by a high glucose [13][14].At present, Dendrobium o cinale had been made into eye drops for the treatment of keratoxerosis. The clinical and animal study con rmed that Dendrobium o cinale can play a protective role in diabetic keratopathy by inhibiting aldose reductase activity and promoting the expression of laminin [15].This is consistent with our research results, which showed that the treatment of Supplementing Qi and nourishing Yin could obviously protect the corneal nerve bers from high glucose, restore corneal sensitivity,blink re ex and ocular surface homeostasis, and reduce the clinical bad symptoms of dry eye decrease.
Normal corneal endothelial cells have a hexagonal structure with almost unchanged cell shape and size.This structure maintains the surface tension and constancy of the cells. Once destroyed, it will affect the cell function. In this study, we found that the morphology of endothelial cells in the treatment group were not obvious change, while in the control group, the cell morphology changed obviously and lost normal hexagon,and the cell stability was destroyed. This change was related to aging, hypoxic injury (diabetes) and stress [16].Goldstein found that the morphology of endothelial cell was signi cant abnormality in both type 1 and type 2 diabetes, and a more signi cant deterioration in type 1 diabetes [17]. In this study, the morphology of endothelial cells in the treatment group was only slightly damaged, and the hexagon was well maintained, which indicated that the treatment of Supplementing Qi and nourishing Yin could protect corneal endothelial cells from the double damage of diabetes and dry eye disease, and restore the function of corneal inner barrier. The balance of uid in corneal stroma is regulated by multiple tight junctions of endothelial cells and the action of Na + /K + ATPase pump. In this study, we found that corneal thickness increase signi cantly in the control group.Considering that the internal barrier function of the cornea was destroyed to make the uid in matrix unbalance [18].The function of mitochondria was destroyed, ATP production was decreased in corneal endothelial cells and epithelial cells,the permeability of endothelial cells was increased,these pathological changes destroyed the tight junction and Na + /K + ATPase pump function and aggravated matrix edema [19].And these abnormal changes were related to the age of diabetic patients [20].The corneal thickness was signi cantly lower in the treatment group,indicating that the function of corneal internal and external barrier was slightly damaged by dry eye disease and hyperglycemia, and the discomfort was further alleviated and visual function were further improved.
Therefore, we concluded that Shumu Decoction(Yiqi Yangyin Chinese medicine) could alleviate the uncomfortable symptoms of patients with diabetic dry eye by delaying the loss of corneal cells and nerve bers and restoring the normal function of cornea.

Conclusion
Shumu Decoction takes human as a whole, focuses on diabetic keratopathy, clari es the protective mechanism of cornea, and clari es the mechanism of Shumu Decoction in treating diabetic dry eye.

List Of Abbreviations
Traditional Chinese Medicine: TCM

Declarations
Ethics approval and consent to participate: