Home | Register | Login | Inquiries | Alerts | Sitemap |  


Advanced Search
JKM > Volume 34(3); 2013 > Article
Jong bin, Jung, and Sik: Effects of Bogijetong-Tang on Diabetic-peripheral Neuropathy Induced by Streptozotocin in the Mouse

Abstract

Objectives:

Previous reports have shown that Bogijetong-Tang (BJT) is effective in peripheral neuropathy induced by taxol and crush injury. In this study, we researched the effects of BJT on diabetic neuropathy induced by STZ in the mouse.

Methods:

We performed both in vitro and in vivo experiments to verify the effects of BJT on diabetic neuropathy induced by STZ in mice. Changes in axonal recovery were observed with immunofluorescence staining using NF-200, Hoechst33258, S100β, caspase 3 and anti-cdc2. Proliferation and degeneration of Schwann cells were investigated by immunofluorescence staining and western blot analyses.

Results:

BJT showed considerable effects on neurite outgrowth and axonal regeneration in diabetic neuropathy. BJT contributed to the creation of NF-200, GAP-43, Cdc2, phosphovimentin, β1, active β1, β3 integrin, phospho-Erk1/2 protein.

Conclusions:

Through this study, we found that BJT is effective for enhanced axonal regeneration via dynamic regulation of regeneration-associated proteins. Therefore, BJT had a pharmaceutical property enhancing recovery of peripheral nerves induced by diabetic neuropathy and could be a candidate for drug development after more research.

Fig. 1.
Pattern of neurite outgrowth of cultured DRG neurons.
DRG sensory neurons were treated with glucose and BJT at 0.3mg/ml or 0.5mg/ml for 24hr. Cells were visualized by immunofluorescence staining for NF-200 protein (blue). (A) Representative images of immunostained cells. (B) Quantification of neurite outgrowth. n=4 ***:p<0.001 (vs normal group) †: p<0.05, ††: p<0.01 (vs control group). Scale bar in (A): 50μm.
Normal : non-treated group.
Control : glucose(30mM) + saline
BJT0.3 : glucose(30mM) + BJT(0.3mg/ml)
BJT0.5 : glucose(30mM) + BJT(0.5mg/ml)
jkm-34-3-126-11f1.tif
Fig. 2.
Distribution of DRG neurons and non-neuronal cells of cultured DRG tissue.
Cultured DRG cells including sensory neurons and non-neuronal cells were identified by NF-200 immunostaining (green) and Hoechst nuclear staining (blue). Merged images show their proximity in distribution in culture. Scale bar: 50μm.
Control : glucose(30mM) + saline
BJT0.3 : glucose(30mM) + BJT(0.3mg/ml)
BJT0.5 : glucose(30mM) + BJT(0.5mg/ml)
jkm-34-3-126-11f2.tif
Fig. 3.
Immunofluorescence staining analysis of NF-200 protein signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for NF-200 protein (in green).
Scale bar: 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f3.tif
Fig. 4.
Immunofluorescence staining analysis of GAP-43 protein signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for GAP-43 protein (in red).
Scale bar: 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f4.tif
Fig. 5.
Western blot analysis of GAP-43 in protein samples from the sciatic nerves after different treatments.
GAP-43 protein was identified as a single band at 43 kDa.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f5.tif
Fig. 6.
Hoechst nuclear staining of sciatic nerve sections. Longitudinal nerve sections were used for Hoechst nuclear staining, and individual nuclei were detected (in blue).
(A) Representative images in nerve sections for individual experimental groups. (B) Quantitative comparison of the number of nuclei among individual experimental groups. n=4, error bar: SEM. *P<0.05, compared to normal group. Scale bar in (A): 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f6.tif
Fig. 7.
Western blot analysis of Cdc2 in protein samples from the sciatic nerves after different treatments.
Cdc2 protein was identified as a single band at 34 kDa.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f7.tif
Fig. 8.
Immunofluorescence staining analysis of Cdc2 protein signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining. (A) Representative fluorescence views for NF-200 protein (green). (B) Double immunofluorescence staining of Cdc2 and S100 β protein signals. Transverse nerve sections from the injured sciatic nerves were used to examine colocalization of two proteins. Scale bars in (A) and (B): 100μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f8.tif
Fig. 9.
Western blot analysis of phospho-vimentin in protein samples from the sciatic nerves after different treatments. Phospho-vimentin was identified as a single band at 57 kDa.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f9.tif
Fig. 10.
Immunofluorescence staining analysis of phospho-vimentin signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for phospho-vimentin (in green). (A) Representative fluorescence staining images. (B) Merged images of phospho-vimentin with Hoechst-stained nuclei. Scale bar in (A, B): 100μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f10.tif
Fig. 11.
Western blot analysis of phospho-Erk1/2 in protein samples from the sciatic nerves after different treatments
Phospho-Erk1/2 was identified as double bands at 42 and 44kDa (arrows).
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f11.tif
Fig. 12.
Immunofluorescence staining analysis of phospho-Erk1/2 signals in the longitudinal sciatic nerve sections
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for phospho-Erk1/2 (in red). (A) Representative fluorescence staining images. (B) Merged images of phospho-Erk1/2 with Hoechst-stained nuclei.
Scale bar in (A): 100μm, which also applies to (B).
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f12.tif
Fig. 13.
Double Immunofluorescence staining of phospho-Erk1/2 (red) and phospho-vimentin (green).
The images shown here are prepared from the nerve of the animal treated with both STZ and BJT. Scale bar: 100μm.
jkm-34-3-126-11f13.tif
Fig. 14.
Western blot analysis of β1 integrin in protein samples from the sciatic nerves after different treatments.
β1 integrin was identified as double bands at 130 kDa.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f14.tif
Fig. 15.
Immunofluorescence staining analysis of β1 integrin signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for β1 integrin (in red). (A) Representative fluorescence staining images. (B) Merged images of β1 integrin with Hoechst-stained nuclei. Scale bar in (A, B): 100μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f15.tif
Fig. 16.
Western blot analysis of active β1 integrin in protein samples from the sciatic nerves after different treatments.
Active β1 integrin was identified as double bands at 130 kDa.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f16.tif
Fig. 17.
Immunofluorescence staining analysis of active form of β1 integrin signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for active β1 integrin (in green). (A) Representative fluorescence staining images. (B) Double immunofluorescence staining of the nerves treated with STZ and BJT with total (red) and active β1 integrin proteins (green). Scale bar in (A, B): 100μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f17.tif
Fig. 18.
Immunofluorescence staining analysis of β 3 integrin signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for β3 integrin (in red). Scale bar: 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f18.tif
Fig. 19.
Double stained images of β 3 integrin in the nerves treated with STZ and BJT.
(A) Merged images of β3 integrin with Hoechst-stained nuclei. (B) Double immunofluorescence staining with β3 integrin proteins (red) and active β1 integrin (green). Scale bar in (A, B): 100μm.
jkm-34-3-126-11f19.tif
Fig. 20.
Immunofluorescence staining analysis of phospho-Smad signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for phospho-Smad (in red).
Scale bar: 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f20.tif
Fig. 21.
Immunofluorescence staining analysis of caspase 3 signals in the longitudinal sciatic nerve sections.
Following treatments of STZ and BJT, sciatic nerve sections were used for immunofluorescence staining for caspase 3 (in red). (A) Representative fluorescence staining images. (B) Merged images of β1 integrin with Hoechst-stained nuclei. Scale bar in (A, B): 200μm.
Normal : non-treated group.
Control : STZ(200mg/kg) + saline
BJT : STZ(200mg/kg) + BJT(400mg/kg)
jkm-34-3-126-11f21.tif
Table 1.
Prescription of Bogijetong-tang(BJT)
Scientific name Amount (g)
Astragali Radix 30
Ginseng Radix 4
Angelicae Gigantis Radix 7.5
Rehmanniae Radix Preparat 10
Cnidii Rhizoma 5
Paeoniae Radix Rubra 7.5
Salviae Miltiorrhizae Radix 12
Persicae Semen 7.5
Carthami Flos 7.5
Spatholobi Caulis 12
Epimedii Herba 10
Lumbricus 5
Puerariae Radix 8
Cibotii Rhizoma 8
Albiziae Cortex 12
Uncariae Ramulus et Uncus 12
Chaenomelis Fructus 8
Ostrae Concha 12

Total amount 178

References

1.. The Whole Country College Korean Medicine, Department of Singye Internal Medicine. Singye Internal Medicine. Seoul: Koonja publishing co.;2011. p. 310


2.. Shaw JE, Zimmet PZ. The epidemiology of diabetic neuropathy. Diabetes. 1999; 7:245–52.


3.. Kathleen M, Halat , Cathi E, Dennehy , Pharm D. Botanicals and Dietary Supplements in Diabetic Peripheral Neuropathy. J Am Board Fam Pract. 2003; 16:1. 47–57.
crossref pmid

4.. Kinoshita JH, Nishimura C. The involvement of aldose reductase in diabetic complications. Diabetes Metab. 1988; 4:323–37.
crossref pmid

5.. Kaul CL, Ramarao P. The role of aldose reductase inhibitions in diabetic complications: recent trends. Methods Find Exp Clin Phamacol. 2001; 23:465–75.
crossref pmid

6.. Green DA, Lattimer SA, Sima AF. Sorbitol, phosphoinositides and sodium-potassium ATPase in the pathogenesis of diabetic complication. The New England Journal of medicine. 1987; 316:10. 599–606.
pmid

7.. Dyck PJ. Detection, characterization and staging of polyneuropathy: assessed in diabetes. Muscle & Nerve. 1988; 11:1. 21–32.
crossref pmid

8.. Kim JM, Cho CS, Kim CJ. Overview of Diabetic Peripheral Neuropathy and Need for Therapeutic Strategy using Traditional Korean Medicine. J Korean Oriental Med. 2009; 30:5. 127–36.


9.. Chen YS, Wu CH, YAO CH, Chen CT. Ginsenoside Rb-1 enhance peripheral nerve regeneration across wide gaps in silicone rubber chambers. Int J Artif Organ. 2002; Nov. 25:11. 1103–8.
pmid

10.. Korean Diabetes Association, Group for Research on Neuropathy. Diabetic Neuropathy. Seoul: Gold Gihoek;2006. p. 172–3.


11.. Kang SB. 2 case of Diabetic Neuropathy Treatments. J Korean Oriental Med. 1992; 13:2. 22–5.


12.. Park SW, Kang JK, Moon SK, Ko CN. Two experiences of the treatment for diabetic peripheral neuropathy. Journal of Korean Oriental Chronic Disease. 1997; 3:1. 251–8.


13.. Cho KH, Jung S, Lee KJ. A Case of Yukmijihwanghwan's effect on Diabetic neuropathy. Korean J. Orient. Int. Med. 1999; 20:1. 286–90.


14.. Kwon YK, Choi KR, Lee JS, Lee BC, Ahn YM, Ahn SY, et al. Two Cases of Diabetic Peripheral Polyneuropathy Improved by Bogan-tang. J Korean Oriental Med. 2002; 23:1. 170–7.


15.. Park SK, Kwon EH, Shin HC, Kang SB. One case of Diabetic Peripheral Polyneuropathy Improved by Binsosan-gamibang. J Korean Oriental Med. 2005; 26:4. 935–40.


16.. Choi HS, Cho CS, Kim CJ. Clinical Study on Two Cases of In patients with Diabetic Peripheral Neuropathy. Daejeon University Institute of korean medicine thesis Collection. 2004; 13:2. 251–8.


17.. Kim JM, Cho CS, Kim CJ. Clinical Study of 8 Diabetic Patients with Paresthesia. Korean J Orient Int Med. 2010; 31:2. 184–91.


18.. An SH. Effects of Bogijetong-tang treatment on animal model of peripheral neuropathy induced by Taxol and crush injury. Doctoral Dissertation. Daejeon: Daejeon Univ;2012.


19.. Banker G, Goslin K. Culturing nerve cells. MIT press;2002.


20.. Felderman EL, Russell JW, Sullivan KA, Golovoy D. New insights into the pathogenesis of diabetic neuropathy. Curr Opin Neurol. 1999; 12:553–63.
crossref pmid

21.. Verrotti A, Giuva PT, Morgese G, Chiarelli F. New trends in the etiopathogenesis of diabetic peripheral neuropathy. J Child Neurol. 2001; 16:389–94.
crossref pmid

22.. Stevens MJ, Felderman EL, Greene DA. The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. Diabet Med. 1995; 12:566–79.
crossref pmid

23.. Greene DA, Lewis RA, Lattimer SA, Brown MJ. Selective effects of myo-inositol administration on sciatic and tibial motor nerve conduction parameters in the streptozocin-diabetic rat. Diabetes. 1982; 31:573–8.
crossref pmid

24.. Greene DA, Sima AA, Stevens MJ, Felderman EL, Lattimer SA. Complication: neuropathy, pathogenetic consideration. Diabetes Cares. 1992; 15:1902–25.
crossref pmid

25.. Brownlee M, Vlassara H, Cerami A. Inhibition of heparin-catalyzed human antithrombin activity by nonenzymatic glycosylation. Possible role in fibrin deposition in diabetes. Diabetes. 1984; 33:532–5.
crossref pmid

26.. Tilton RG, Chang K, Nyengaard JR, Van den Enden M, Ido Y, Williamson JR. Inhibition of sorbitol dehydrogenase. Effects on vascular and neural dysfunction in streptozotocin induced diabetic rats. Diabetes. 1995; 44:234–42.
crossref pmid

27.. Nagamatsu M, Nickander KK, Schmelzer JD, Raya A, Wittrock DA, Tritschler H, et al. Lipoic acid improves nerve blood flow, reduces oxidative stress, and improves distal nerve conduction in experimental diabetic neuropathy. Diabetes Care. 1995; 18:1160–7.
crossref pmid

28.. Karasu C, Dewhurst M, Stevens EJ, Tomlinson DR. Effects of antioxidant treatment on sciatic nerve dysfunction in streptozotocin-diabetic rats; comparison with essential fatty acids. Diabetologia. 1995; 38:129–134.
crossref pmid

29.. Hotta N, Koh N, Sakakibara F, Nakamura J, Hamada Y, Wakao T, et al. Prevention of abnormalities in motor nerve conduction and nerve bloodflow by a prostacyclin analog, beraprost sodium, in streptozotocin-induced diabetic rats. Prostaglandins. 1995; 49:339–49.
pmid

30.. Cameron NE, Cotter MA. Metabolic and vascular factors in the pathogenesis of diabetic neuropathy. Diabetes. 1997; 2:S31–7.
crossref pmid

31.. Hounsom L, Horrobin DF, Tritschler H, Corder R, Tomlinson DR. A lipoic acid-gamma linolenic acid conjugate ie effective against multiple indices og experimental diabetic neuropathy. Diabetologia. 1998; 41:839–43.
crossref pmid

32.. A society for the research of Neuropathy in Korean Diabetes Association. Clinical practice of diabetic neuropathy. 2nd ed. Seoul: Korean Diabetes Association;2007. p. 1–48.


33.. Doo HK. Internal medicine of kidney in oriental medicine. Seoul: Institute of oriental medicine;1991. p. 969–76.


Editorial office contact information
3F, #26-27 Gayang-dong, Gangseo-gu Seoul, 157-200 Seoul, Korea
The Society of Korean Medicine
Tel : +82-2-2658-3627   Fax : +82-2-2658-3631   E-mail : skom1953@daum.net
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Developed in M2PI